Antibody adjuvant conjugates

ABSTRACT

The invention provides an immunoconjugate comprising an antibody construct which includes an antigen binding domain and an Fc domain, an adjuvant moiety, and a linker, wherein each adjuvant moiety is covalently bonded to the antibody via the linker. Methods for treating cancer with the immunoconjugates of the invention are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. patentapplication Ser. No. 16/140,309, filed Sep. 24, 2018, which is acontinuation of International Patent Application No. PCT/US2017/041268,filed on Jul. 7, 2017, which claims the benefit of U.S. ProvisionalApplication 62/359,626, filed on Jul. 7, 2016, U.S. ProvisionalApplication 62/359,627, filed on Jul. 7, 2016, U.S. ProvisionalApplication 62/432,530, filed on Dec. 9, 2016, U.S. ProvisionalApplication 62/433,742, filed on Dec. 13, 2016, U.S. ProvisionalApplication 62/522,623, filed on Jun. 20, 2017, and U.S. ProvisionalApplication 62/526,306, filed on Jun. 28, 2017, the disclosures of whichare incorporated herein by reference in their entireties for allpurposes.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 13 kilobyte file named“STAN-1340CON-SeqList-ST25.txt,” created Mar. 12, 2020.

BACKGROUND OF THE INVENTION

It is now well appreciated that tumor growth necessitates theacquisition of mutations that facilitate immune evasion. Even so,tumorigenesis results in the accumulation of mutated antigens, orneoantigens, that are readily recognized by the host immune systemfollowing ex vivo stimulation. Why and how the immune system fails torecognize neoantigens are beginning to be elucidated. Groundbreakingstudies by Carmi et al. (Nature, 521: 99-104 (2015)) have indicated thatimmune ignorance can be overcome by delivering neoantigens to activateddendritic cells via antibody-tumor immune complexes. In these studies,simultaneous delivery of tumor binding antibodies and dendritic celladjuvants via intratumoral injections resulted in robust anti-tumorimmunity. New compositions and methods for the delivery of antibodiesand dendritic cell adjuvants are needed in order to reach inaccessibletumors and to expand treatment options for cancer patients and othersubjects

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides an immunoconjugate comprising(a) an antibody construct comprising (i) an antigen binding domain and(ii) an Fc domain, (b) an adjuvant moiety, and (c) a linker, whereineach adjuvant moiety is covalently bonded to the antibody construct viathe linker.

In some embodiments, the immunoconjugate has a structure according toFormula I:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodyconstruct; A is an unmodified amino acid sidechain in the antibodyconstruct or a modified amino acid sidechain in the antibody construct;Z is a linking moiety; Adj is an adjuvant moiety; and subscript r is aninteger from 1 to 10.

In a related aspect, the invention provides a composition comprising aplurality of immunoconjugates as described herein.

In another aspect, the invention provides a method for treating cancer.The method includes administering a therapeutically effective amount ofan immunoconjugate according to the invention to a subject in needthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. According tocommon practice, the various features of the drawings are not to-scale.On the contrary, the dimensions of the various features are arbitrarilyexpanded or reduced for clarity.

FIG. 1 shows that functionalized adjuvant is a potent inducer of myeloidcell activation. Peripheral blood antigen presenting cells (APCs) werestimulated with 10-fold serial dilutions of R848, Compound 2 or acontrol TLR agonist at 37° C. After 18 hours, cells were analyzed viaflow cytometry. Data are presented as median fluorescence intensity ofeach indicated marker; n=3.

FIG. 2 shows that functionalized adjuvants maintain TLR agonistactivity. HEK293 cells were co-transfected with human TLR7 or TLR8 (toptwo panels) or murine TLR7 (bottom panel) and an inducible secretedembryonic alkaline phosphatase reporter gene under the control of theIFN-β minimal promoter fused to NF-κB and AP-1 binding sites. Cells weresubsequently incubated with 2-fold serial dilutions of the indicatedadjuvant for 12 hours at 37° C. Activity was measured byspectrophotometry (OD 650 nm) following addition of alkaline phosphatasesubstrate.

FIG. 3 shows the analysis of adjuvant linker compounds via liquidchromatography-mass spectrometry (LC-MS).

FIG. 4 shows that antibody-adjuvant conjugates are superior at elicitingAPC activation, compared to unconjugated antibody and adjuvant, asindicated by expression of CD40, CD86 and HLA-DR. Human APCs werestimulated with Rituximab-SATA-SMCC-Compound 1 (conjugated), Rituximabalone (Ab), Compound 1 alone or Rituximab+Compound 1 (Mixture) in thepresence of CFSE-labeled CD19+ tumor cells. After 18 hours, CD19− humanAPCs were analyzed via flow cytometry; n=3. P-values ≤0.05 depicted by*, P-values ≤0.01 depicted by **, P-values ≤0.001 depicted by ***,P-values ≤0.0001 depicted by ****

FIG. 5 shows that antibody-adjuvant conjugates induce lower levels ofPD-L1 expression on human APCs, compared to unconjugated antibody andadjuvant. Human APCs were stimulated with Rituximab-SATA-SMCC-Compound 1(conjugated), Rituximab alone (Ab), Compound 1 alone orRituximab+Compound 1 (Mixture) in the presence of CFSE-labeled CD19tumor cells. After 18 hours, CD19⁻ human APCs were analyzed via flowcytometry; n=3. P-values ≤0.05 depicted by *, P-values ≤0.01 depicted by**, P-values ≤0.001 depicted by ***, P-values ≤0.0001 depicted by ****.

FIG. 6 shows that antibody-adjuvant conjugates elicit DCdifferentiation. Human APCs that were ˜95% monocytes were stimulatedwith 2-fold serial dilutions of Rituximab-SATA-SMCC-Compound 1(conjugated), Rituximab alone (Ab), Compound 1 alone orRituximab+Compound 1 (Mixture) in the presence of CFSE-labeled tumorcells. After 18 hours, CD19⁻ human APCs were analyzed via flowcytometry; n=3. P-values ≤0.05 depicted by *, P-values ≤0.01 depicted by**, P-values ≤0.001 depicted by ***, P-values ≤0.0001 depicted by ****

FIG. 7 shows that antibody-adjuvant conjugates are superior to mixturesof unconjugated antibody and adjuvant for eliciting the secretion ofproinflammatory cytokines from human APCs. Human APCs were stimulatedwith 2-fold serial dilutions of Rituximab-SATA-SMCC-Compound 1(conjugated), Rituximab alone (Ab), Compound 1 alone orRituximab+Compound 1 (Mixture) in the presence of fixed, CFSE-labeledtumor cells. After 18 hours, cell free supernatants were analyzed forcytokine secretion via cytokine bead arrays; n=3. P-values ≤0.05depicted by *, P-values ≤0.01 depicted by **, P-values ≤0.001 depictedby ***, P-values ≤0.0001 depicted by ****

FIG. 8A shows that immunoconjugates with cleavable linkers elicit APCactivation and DC differentiation. Human APCs that were ˜95% monocyteswere stimulated with 2-fold serial dilutions ofRituximab-SATA-SPDP-Compound 1 (Conjugated, cleavable), Rituximab alone(Ab), Compound 1 alone or Rituximab+Compound 1 (Mixture) in the presenceof CFSE-labeled tumor cells. The immunoconjugate (AAC—cleavable) had adrug to antibody ratio (DAR) of 1.4 as confirmed by MALDI-TOF. After 18hours, CD19− human APCs (CD14 and CD123) were analyzed via flowcytometry; n=3.

FIG. 8B shows that immunoconjugates (AACs) with cleavable linkers elicitAPC activation and DC differentiation. Human APCs that were ˜95%monocytes were stimulated with 2-fold serial dilutions ofRituximab-SATA-SPDP-Compound 1 (Conjugated, cleavable), Rituximab alone(Ab), Compound 1 alone or Rituximab+Compound 1 (Mixture) in the presenceof CFSE-labeled tumor cells. The immunoconjugates (AAC—Cleavable) had adrug to antibody ratio (DAR) of 1.4 as confirmed by MALDI-TOF. After 18hours, CD19− human APCs (CD16 and CD163) were analyzed via flowcytometry; n=3.

FIG. 8C shows that immunoconjugates with cleavable linkers elicit APCactivation and DC differentiation. Human APCs that were ˜95% monocyteswere stimulated with 2-fold serial dilutions ofRituximab-SATA-SPDP-Compound 1 (Conjugated, cleavable), Rituximab alone(Ab), Compound 1 alone or Rituximab+Compound 1 (Mixture) in the presenceof CFSE-labeled tumor cells. Immunoconjugates (AAC—Cleavable) had a drugto antibody ratio (DAR) of 1.4 as confirmed by MALDI-TOF. After 18hours, CD19− human APCs (CD40 and PDL1) were analyzed via flowcytometry; n=3.

FIG. 9A shows that antibody-adjuvant conjugates reduce tumors in vivo.C57BL/6 mice with B 16F10 tumors in the right flank were injectedintratumorally with PBS (Untreated), uGP75+Compound 1 (Mixture) oruGP75-SATA-SMCC-Compound 1 (uGP75-immunoconjugate).

FIG. 9B shows that αGP75-immunoconjugate reduces tumors in vivo whenadministered via intratumoral (IT) or intravenous (IV) injection.

FIG. 10A shows the analysis of ipilimumab via LC-MS.

FIG. 10B shows that ipilimumab-adjuvant (Ipilimumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatedipilimumab, as indicated by expression of HLA-DR.

FIG. 10C shows that ipilimumab-adjuvant (Ipilimumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatedipilimumab, as indicated by expression of CD14.

FIG. 10D shows that ipilimumab-adjuvant (Ipilimumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatedipilimumab, as indicated by expression of CD40.

FIG. 10E shows that ipilimumab-adjuvant (Ipilimumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatedipilimumab, as indicated by expression of CD86.

FIG. 11A shows the analysis of pembrolizumab via LC-MS.

FIG. 11B shows that pembrolizumab-adjuvant (Pembrolizumab Boltbody)conjugates are superior at eliciting APC activation, compared tounconjugated pembrolizumab, as indicated by expression of HLA-DR.

FIG. 11C shows that pembrolizumab-adjuvant (Pembrolizumab Boltbody)conjugates are superior at eliciting APC activation, compared tounconjugated pembrolizumab, as indicated by expression of CD 14.

FIG. 11D shows that pembrolizumab-adjuvant (Pembrolizumab Boltbody)conjugates are superior at eliciting APC activation, compared tounconjugated pembrolizumab, as indicated by expression of CD40.

FIG. 11E shows that pembrolizumab-adjuvant (Pembrolizumab Boltbody)conjugates are superior at eliciting APC activation, compared tounconjugated pembrolizumab, as indicated by expression of CD86.

FIG. 12A shows the analysis of nivolumab via LC-MS.

FIG. 12B shows that nivolumab-adjuvant (Nivolumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatednivolumab, as indicated by expression of HLA-DR.

FIG. 12C shows that nivolumab-adjuvant (Nivolumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatednivolumab, as indicated by expression of CD14.

FIG. 12D shows that nivolumab-adjuvant (Nivolumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatednivolumab, as indicated by expression of CD40.

FIG. 12E shows that nivolumab-adjuvant (Nivolumab Boltbody) conjugatesare superior at eliciting APC activation, compared to unconjugatednivolumab, as indicated by expression of CD86.

FIG. 13A shows the analysis of atezolizumab via LC-MS.

FIG. 13B shows that atezolizumab-adjuvant (Atezolizumab Boltbody)conjugates are superior at eliciting APC activation, compared tounconjugated atezolizumab, as indicated by expression of HLA-DR.

FIG. 13C shows that atezolizumab-adjuvant (Atezolizumab Boltbody)conjugates are superior at eliciting APC activation, compared tounconjugated atezolizumab, as indicated by expression of CD14.

FIG. 13D shows that atezolizumab-adjuvant (Atezolizumab Boltbody)conjugates are superior at eliciting APC activation, compared tounconjugated atezolizumab, as indicated by expression of CD40.

FIG. 13E shows that the level of activation of atezolizumab-adjuvant(Atezolizumab Boltbody) conjugates, as indicated by expression of CD86.

FIG. 14A shows that atezolizumab immunoconjugate (Atezolizumab IgG1 NQBoltbody)-differentiated cells secrete higher amounts of TNFα thanatezolizumab-differentiated cells.

FIG. 14B shows that atezolizumab immunoconjugate (Atezolizumab IgG1 NQBoltbody)-differentiated cells secrete higher amounts of IL-10 thanatezolizumab-differentiated cells.

FIG. 15A shows that nivolumab immunoconjugate (Nivolumab IgG4Boltbody)-differentiated cells secrete higher amounts of TNFα thannivolumab-differentiated cells.

FIG. 15B shows that nivolumab immunoconjugate (Nivolumab IgG4Boltbody)-differentiated cells secrete higher amounts of IL-10 thannivolumab-differentiated cells.

FIG. 16A shows that pembrolizumab immunoconjugate (PembrolizumabBoltbody)-differentiated cells secrete higher amounts of TNFα thanpembrolizumab-differentiated cells.

FIG. 16B shows that pembrolizumab immunoconjugate (PembrolizumabBoltbody)-differentiated cells secrete higher amounts of IL-1β thanpembrolizumab-differentiated cells.

FIG. 17 shows the analysis of pembrolizumab-adjuvant conjugates viaLC-MS.

FIG. 18 shows the analysis of nivolumab-adjuvant conjugates via LC-MS.

FIG. 19 shows the analysis of atezolizumab-adjuvant conjugates viaLC-MS.

FIG. 20 shows that ipilimumab immunoconjugate (IpilimumabBoltbody)-differentiated cells secret higher amounts of TNFα thanipilimumab-differentiated cells.

FIG. 21 shows that Dectin-2 immunoconjugate-differentiated cells secretehigher amounts of TNFα, IL-6, and IL-12p70 than cells exposed toequivalent amounts of the unconjugated components. The line that issignificantly higher than the x-axis for each cytokine is theanti-Dectin-2 immunoconjugate (anti-Dectin-2-Cmpd1 (antibody conjugatedwith adjuvant Compound 1)). There are three lines along the x-axis,which are not visible, which show that the anti-Dectin-2 antibody alone,and the adjuvant Compound 1 alone, and the anti-Dectin 2 antibody andadjuvant Compound 1 mixture (anti-Dectin-2+Cmpd1 mixture, unconjugated),failed to produce any cytokine response. The line which is barely abovethe x-axis for IL-6 represents a control antibody, an ACC with Compound1 as the adjuvant and a rat IgG2a isotype control antibody (labeled“Iso-Cmpd 1” in FIG. 21). In the TNFα and IL-12p70 graphs, the Iso-Cmpd1 line is not visible as it is along the x-axis.

FIG. 22A shows the structure of adjuvant CL264 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, the terminal carboxylic acid of the adjuvant.

FIG. 22B shows the structure of adjuvant CL401 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, the primary amine of the adjuvant.

FIG. 22C shows the structure of adjuvant CL413 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, the first lysine residue of the adjuvant.

FIG. 22D shows the structure of adjuvant CL413 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, the second lysine residue of the adjuvant.

FIG. 22E shows the structure of adjuvant CL413 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, the third lysine residue of the adjuvant.

FIG. 22F shows the structure of adjuvant CL413 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, the fourth lysine residue of the adjuvant.

FIG. 22G shows the structure of adjuvant CL413 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, the primary amine of the adjuvant.

FIG. 22H shows the structure of adjuvant CL419 and the circles indicatepositions on the adjuvant where it could be conjugated to the linker,specifically, the amines of the adjuvant (terminal amine in the top partof FIG. 22H and secondary amine in the bottom part of FIG. 22H).

FIG. 22I shows the structure of adjuvant CL553 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, a secondary amine of the adjuvant.

FIG. 22J shows the structure of adjuvant CL553 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, another secondary amine of the adjuvant.

FIG. 22K shows the structure of adjuvant CL553 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, a primary amine of the adjuvant.

FIG. 22L shows the structure of adjuvant CL553 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, another secondary amine of the adjuvant.

FIG. 22M shows the structure of adjuvant CL553 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, another secondary amine of the adjuvant.

FIG. 22N shows the structure of adjuvant CL553 and the circle indicatesa position on the adjuvant where it could be conjugated to the linker,specifically, another secondary amine of the adjuvant.

FIG. 22O shows the structure of adjuvant CL572 and the circles indicatepositions on the adjuvant where it could be conjugated to the linker,specifically, the primary amine (top part of FIG. 22O) and the carbonyl(bottom part of FIG. 22O).

FIG. 22P shows the structure of adjuvant Pam2CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the terminal carboxylic acid of the adjuvant.

FIG. 22Q shows the structure of adjuvant Pam2CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the terminal thiol of the adjuvant.

FIG. 22R shows the structure of adjuvant Pam2CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the second lysine residue of the adjuvant.

FIG. 22S shows the structure of adjuvant Pam2CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the third lysine residue of the adjuvant.

FIG. 22T shows the structure of adjuvant Pam2CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the terminal lysine residue of the adjuvant.

FIG. 22U shows the structure of adjuvant Pam3CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the terminal carboxylic acid of the adjuvant.

FIG. 22V shows the structure of adjuvant Pam3CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the terminal thiol of the adjuvant.

FIG. 22W shows the structure of adjuvant Pam3CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the second lysine residue of the adjuvant.

FIG. 22X shows the structure of adjuvant Pam3CSK4 and the circleindicates a position on the adjuvant where it could be conjugated to thelinker, specifically, the third lysine residue of the adjuvant.

FIG. 23A shows αCLEC5A immunoconjugate-differentiated cells secretehigher amounts of IL-6 than cells exposed to equivalent amounts of theunconjugated components. The line that is significantly higher than thex-axis for each cytokine is αCLEC5A immunoconjugate (αCLEC5A antibodyconjugated with adjuvant Compound 1). The line that is along the x-axisshows that a mixture of αCLEC5A antibody and adjuvant Compound 1(unconjugated) failed to produce any cytokine response. The line betweenthe x-axis and the αCLEC5A immunoconjugate line represents a controlconjugate, rat IgG2a isotype conjugated to Compound 1.

FIG. 23B shows αCLEC5A immunoconjugate-differentiated cells secretehigher amounts of IL-12p40 than cells exposed to equivalent amounts ofthe unconjugated components. The line that is significantly higher thanthe x-axis for each cytokine is αCLEC5A immunoconjugate (αCLEC5A-Cmpd1AAC). The line that is along the x-axis shows that a mixture of αCLEC5Aantibody and adjuvant Compound 1 (unconjugated; αCLEC5A-Cmpd1 Mixture)failed to produce any cytokine response. The line between the x-axis andthe αCLEC5A immunoconjugate line represents a control conjugate, ratIgG2a isotype conjugated to Compound 1.

FIG. 23C shows αCLEC5A immunoconjugate-differentiated cells secretehigher amounts of IL-12p70 than cells exposed to equivalent amounts ofthe unconjugated components. The line that is significantly higher thanthe x-axis for each cytokine is αCLEC5A immunoconjugate (αCLEC5Aantibody conjugated with adjuvant Compound 1). The line that is alongthe x-axis shows that a mixture of αCLEC5A antibody and adjuvantCompound 1 (unconjugated) failed to produce any cytokine response. Theline between the x-axis and the αCLEC5A immunoconjugate line representsa control conjugate, rat IgG2a isotype conjugated to Compound 1.

FIG. 23D shows αCLEC5A immunoconjugate-differentiated cells secretehigher amounts of TNFα than cells exposed to equivalent amounts of theunconjugated components. The line that is significantly higher than thex-axis for each cytokine is αCLEC5A immunoconjugate (αCLEC5A antibodyconjugated with adjuvant Compound 1). The line that is along the x-axisshows that a mixture of αCLEC5A antibody and adjuvant Compound 1(unconjugated) failed to produce any cytokine response. The line betweenthe x-axis and the αCLEC5A immunoconjugate line represents a controlconjugate, rat IgG2a isotype conjugated to Compound 1.

FIG. 23E shows the analysis of αCLEC5A immunoconjugate via LC-MS.

FIG. 24 shows increased dendritic cell differentiation with an anti-Her2antibody adjuvant conjugate (αHer2 immunoconjugate, closed circles)linked to the TLR 7/8 agonist Compound 1 as compared to when the sameantibody and adjuvant components (αHer2 and Compound 1, closed squares)are delivered as an unlinked mixture.

FIG. 25 shows increased dendritic cell differentiation with an anti-EGFRantibody adjuvant conjugate (αEGFR immunoconjugate, closed circles)linked to the TLR 7/8 agonist Compound 1 as compared to when the samecomponents (αEGFR and Compound 1, closed squares) are delivered as anunlinked mixture.

FIG. 26 shows that an anti-CD20 antibody conjugated to TLR 7/8 agonistexhibits robust dendritic cell activation, while activation issignificantly reduced in the deglycosylated conjugate.

FIG. 27 compares rituximab and obinutuzumab antibodies conjugated toCompound 1. Obinutuzumab has reduced fucose content as compared torituximab and exhibits increased CD40 upregulation.

FIG. 28 illustrates NK cell activation using an αEGFR immunoconjugatelinked to the TLR 7/8 agonist Compound 1. The immunoconjugate exhibitssignificantly greater NK cell activation as compared to the unconjugatedmixture of αEGFR and Compound 1.

FIG. 29 illustrates robust activation of dendritic cell populations fromperipheral blood mononuclear cells isolated from human subjects with anαEGFR immunoconjugate.

FIG. 30A shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-01 synthesized using the SATA method.

FIG. 30B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-01 synthesized using the ester method.

FIG. 31A shows a size-exclusion chromatography analysis ofimmunoconjugate BB01 synthesized using the SATA method.

FIG. 31B shows a size-exclusion chromatography analysis ofimmunoconjugate BB-01 synthesized using the ester method.

FIG. 32 shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-14 synthesized using the ester method.

FIG. 33 shows a size-exclusion chromatography analysis ofimmunoconjugate BB-14 synthesized using the ester method.

FIG. 34 shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-15 synthesized using the ester method.

FIG. 35 shows a size-exclusion chromatography analysis ofimmunoconjugate BB-15 synthesized using the ester method.

FIG. 36 shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate synthesized using the ester method.

FIG. 37 shows a size-exclusion chromatography analysis ofimmunoconjugate synthesized using the ester method.

FIG. 38A shows BB-01 and BB-17synthesized using the ester method elicitsmyeloid activation as indicated by CD14 downregulation while the controldoes not. CD20 is the unconjugated monoclonal antibody used as acontrol.

FIG. 38B shows BB-01 and BB-17 synthesized using the ester methodelicits myeloid activation as indicated by CD16 downregulation while thecontrol does not. CD20 is the unconjugated monoclonal antibody used as acontrol.

FIG. 38C shows BB-01 and BB-17 synthesized using the ester methodelicits myeloid activation as indicated by CD40 upregulation while thecontrol does not. CD20 is the unconjugated monoclonal antibody used as acontrol.

FIG. 38D shows BB-01 and BB-17 synthesized using the ester methodelicits myeloid activation as indicated by CD86 upregulation while thecontrol does not. CD20 is the unconjugated monoclonal antibody used as acontrol.

FIG. 38E shows BB-01 and BB-17 synthesized using the ester methodelicits myeloid activation as indicated by CD123 upregulation while thecontrol does not. CD20 is the unconjugated monoclonal antibody used as acontrol.

FIG. 38F shows BB-01 and BB-17 synthesized using the ester methodelicits myeloid activation as indicated by Human LeukocyteAntigen-antigen D Related or “HLA-DR” while the control does not. CD20is the unconjugated monoclonal antibody used as a control.

FIG. 39A shows that BB-01 elicits myeloid activation as indicated byCD14 downregulation while comparative IRM1 and IRM2 immunoconjugates donot. CD20 is the unconjugated monoclonal antibody used as a control.

FIG. 39B shows that BB-01 elicits myeloid activation as indicated byCD16 downregulation while comparative IRM1 and IRM2 immunoconjugates donot. CD20 is the unconjugated monoclonal antibody used as a control.

FIG. 39C shows that BB-01 elicits myeloid activation as indicated byCD40 upregulation while comparative IRM1 and IRM2 immunoconjugates donot. CD20 is the unconjugated monoclonal antibody used as a control.

FIG. 39D shows that BB-01 elicits myeloid activation as indicated byCD86 upregulation while comparative IRM1 and IRM2 immunoconjugates donot. CD20 is the unconjugated monoclonal antibody used as a control.

FIG. 39E shows that BB-01 elicits myeloid activation as indicated byCD123 upregulation while comparative IRM1 and IRM2 immunoconjugates donot. CD20 is the unconjugated monoclonal antibody used as a control.

FIG. 39F shows that BB-01 elicits myeloid activation as indicated byHLA-DR upregulation while comparative IRM1 and IRM2 immunoconjugates donot. CD20 is the unconjugated monoclonal antibody used as a control.

FIG. 40A shows that BB-01 elicits cytokine secretion (IL-1β) whilecomparative IRM1 and IRM2 immunoconjugates do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 40B shows that BB-01 elicits cytokine secretion (IL-6) whilecomparative IRM1 and IRM2 immunoconjugates do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 40C shows that BB-01 elicits cytokine secretion (TNFα) whilecomparative IRM1 and IRM2 immunoconjugates do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 41A shows a size-exclusion chromatography analysis ofimmunoconjugate BB-26 synthesized using the ester method.

FIG. 41B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-26 synthesized using the ester method.

FIG. 42A shows a size-exclusion chromatography analysis ofimmunoconjugate BB-27 synthesized using the ester method.

FIG. 42B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-27 synthesized using the ester method.

FIG. 43A shows a size-exclusion chromatography analysis ofimmunoconjugate BB-36 synthesized using the ester method.

FIG. 43B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-36 synthesized using the ester method.

FIG. 44A shows a size-exclusion chromatography analysis of ComparativeConjugate IRM1.

FIG. 44B shows a size-exclusion chromatography analysis of ComparativeConjugate IRM2.

FIG. 44C shows a size-exclusion chromatography analysis of BB-01.

FIG. 45A shows a liquid chromatography-mass spectrometry analysis ofIRM1 conjugate following overnight deglycosylation with PNGase F.

FIG. 45B show a liquid chromatography-mass spectrometry analysis ofBB-01 conjugate following overnight deglycosylation with PNGase F.

FIG. 46A shows a size-exclusion chromatography analysis ofimmunoconjugate BB-45 synthesized using the ester method.

FIG. 46B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-45 synthesized using the ester method.

FIG. 47A shows a size-exclusion chromatography analysis ofimmunoconjugate BB-24 synthesized using the ester method.

FIG. 47B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-24 synthesized using the ester method.

FIG. 48A shows a size-exclusion chromatography analysis ofimmunoconjugate BB-37 synthesized using the ester method.

FIG. 48B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-37 synthesized using the ester method.

FIG. 49A shows a size-exclusion chromatography analysis ofimmunoconjugate BB-42 synthesized using the ester method.

FIG. 49B shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-42 synthesized using the ester method.

FIG. 50 shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-43 synthesized using the ester method.

FIG. 51 shows a liquid chromatography-mass spectrometry analysis ofimmunoconjugate BB-44 synthesized using the ester method.

FIG. 52A shows that BB-14 elicits myeloid activation as indicated byCD14 downregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 52B shows that BB-14 elicits myeloid activation as indicated byCD40 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 52C shows that BB-14 elicits myeloid activation as indicated byCD86 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 52D shows that BB-14 elicits myeloid activation as indicated byHLA-DR upregulation while the control does do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 53A shows that BB-15 elicits myeloid activation as indicated byCD14 downregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 53B shows that BB-15 elicits myeloid activation as indicated byCD40 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 53C shows that BB-15 elicits myeloid activation as indicated byCD86 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 53D shows that BB-27 elicits myeloid activation as indicated byHLA-DR upregulation while the control does do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 54A shows that BB-27 elicits myeloid activation as indicated byCD14 downregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 54B shows that BB-27 elicits myeloid activation as indicated byCD40 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 54C shows that BB-27 elicits myeloid activation as indicated byCD86 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 54D shows that BB-27 elicits myeloid activation as indicated byHLA-DR upregulation while the control does do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 55A shows that BB-45 elicits myeloid activation as indicated byCD14 downregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 55B shows that BB-45 elicits myeloid activation as indicated byCD40 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 55C shows that BB-45 elicits myeloid activation as indicated byCD86 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 55D shows that BB-45 elicits myeloid activation as indicated byHLA-DR upregulation while the control does do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 56A shows that BB-24 elicits myeloid activation as indicated byCD14 downregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 56B shows that BB-24 elicits myeloid activation as indicated byCD40 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 56C shows that BB-24 elicits myeloid activation as indicated byCD86 upregulation while the control does not. CD20 is the unconjugatedmonoclonal antibody used as a control.

FIG. 56D shows that BB-24 elicits myeloid activation as indicated byHLA-DR upregulation while the control does do not. CD20 is theunconjugated monoclonal antibody used as a control.

FIG. 57 shows BB-01 binding to CD20 Toledo cells, which are a cell lineused as a model system for studying non-Hodgkin lymphomas. BB-01 hadstronger binding than the antibodies rituximab or cetuximab.

FIG. 58 shows aEGFR immunoconjugate with Compound 1 (aEGFR Boltbody) wasmore effective than the mixture of antibody and adjuvant at activatingNK cells. PBMCs were activated with the immunoconjugate or the mixturefor 18 hours. NK cells were gated according to lineage negative (CD3,CD19, CD14 negative) and CD56 positive.

FIG. 59 shows the analysis of a comparative immunoconjugate via LC-MS(DG). This comparative conjugate was prepared with trastuzumab and anoncleavable maleimide-PEG4 linker containing a pentafluorophenyl groupwith gardiquimod (see US 2017/0158772, paragraph 0275, description ofimmunoconjugate ATAC3).

FIG. 60 shows the analysis of a comparative immunoconjugate via LC-MS(heavy chain). This comparative conjugate was prepared with trastuzumaband a noncleavable maleimide-PEG4 linker containing a pentafluorophenylgroup with gardiquimod (see US 2017/0158772, paragraph 0275, descriptionof immunoconjugate ATAC3).

FIG. 61 shows the analysis of a comparative immunoconjugate via LC-MS.This comparative conjugate was prepared with trastuzumab and anoncleavable maleimide-PEG4 linker containing a pentafluorophenyl groupwith gardiquimod (see US 2017/0158772, paragraph 0275, description ofimmunoconjugate ATAC3).

FIG. 62 shows the analysis of a comparative immunoconjugate via LC-MS(light chain). This comparative conjugate was prepared with trastuzumaband a noncleavable maleimide-PEG4 linker containing a pentafluorophenylgroup with gardiquimod (see US 2017/0158772, paragraph 0275, descriptionof immunoconjugate ATAC3).

FIG. 63 shows the analysis of a comparative immunoconjugate via LC-MS(DG, heavy chain). This comparative conjugate was prepared withtrastuzumab and a noncleavable maleimide-PEG4 linker containing apentafluorophenyl group with gardiquimod (see US 2017/0158772, paragraph0275, description of immunoconjugate ATAC3).

FIG. 64 shows the analysis of a comparative immunoconjugate via LC-MS(DG, light chain). This comparative conjugate was prepared withtrastuzumab and a noncleavable maleimide-PEG4 linker containing apentafluorophenyl group with gardiquimod (see US 2017/0158772, paragraph0275, description of immunoconjugate ATAC3).

FIG. 65 shows the analysis of a comparative immunoconjugate via LC-MS(DG). This comparative conjugate was prepared with trastuzumab and acleavable valine-citrulline linker containing a PABA group withsuccinamide (see US 2017/0158772, paragraph 0275, description ofimmunoconjugate ATAC2).

FIG. 66 shows the analysis of a comparative immunoconjugate via LC-MS.This comparative conjugate was prepared with trastuzumab and a cleavablevaline-citrulline linker containing a PABA group with succinamide (seeUS 2017/0158772, paragraph 0275, description of immunoconjugate ATAC2).

FIG. 67A shows that the rituximab immunoconjugate produced according tothe BB-01 SATA method (Rituximab Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to unconjugated rituximab(Roche) as well as equimolar concentrations of comparative conjugatesprepared with rituximab and either a valine-citruline-PABC or amaleimide-PEG4 linker, both containing a pentafluorophenyl group withgardiquimod (Rituximab-ATAC2, Rituximab-ATAC3 respectively; US2017/0158772) following 18 hours of stimulation.

FIG. 67B shows that the rituximab immunoconjugate produced according tothe BB-01 SATA method (Rituximab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to unconjugated rituximab(Roche) as well as equimolar concentrations of comparative conjugatesprepared with rituximab and either a valine-citruline-PABC or amaleimide-PEG4 linker, both containing a pentafluorophenyl group withgardiquimod (Rituximab-ATAC2, Rituximab-ATAC3 respectively; US2017/0158772) following 18 hours of stimulation.

FIG. 67C shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method orthe rituximab immunoconjugates according to the methods described in US2017/0158772 following overnight deglycosylation with PNGase F.

FIG. 67D shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method orthe rituximab immunoconjugates according to the methods described in US2017/0158772.

FIG. 67E shows a liquid chromatography-mass spectrometry analysis of theipsilateral heavy-light chain of the rituximab immunoconjugate producedusing a valine-citruline-PABC linker as described in US 2017/0158772following overnight deglycosylation with PNGase F.

FIG. 67F shows a liquid chromatography-mass spectrometry analysis of thelight chain of the rituximab immunoconjugate produced using avaline-citruline-PABC linker as described in US 2017/0158772 followingovernight deglycosylation with PNGase F.

FIG. 67G shows that the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) fails to elicit CD 123 upregulation onmyeloid cells following 18 hours of stimulation. FIG. 67G also showsthat the BB-01 immunoconjugate produced according to the SATA method[Rituximab Boltbody (BB-01)] is superior at eliciting CD123 upregulationas compared to Ritux-ATAC2 and equimolar concentrations of unconjugatedrituximab (Roche).

FIG. 67H shows that the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) fails to elicit CD14 downregulation onmyeloid cells following 18 hours of stimulation. FIG. 67H also showsthat the BB-01 immunoconjugate produced according to the SATA method[Rituximab Boltbody (BB-01)] is superior at eliciting CD14downregulation as compared to Ritux-ATAC2 and equimolar concentrationsof unconjugated rituximab (Roche).

FIG. 67I shows that the BB-01 immunoconjugate produced according to theSATA method [Rituximab Boltbody (BB-01)] is superior at eliciting CD16downregulation on myeloid cells as compared to the rituximab withvaline-citruline-PABC linker immunoconjugate produced according to themethods described in US 2017/0158772 (Ritux-ATAC2) and equimolarconcentrations of unconjugated rituximab (Roche).

FIG. 67J shows that the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) fails to elicit CD40 upregulation on myeloidcells following 18 hours of stimulation. FIG. 67J also shows that theBB-01 immunoconjugate produced according to the SATA method [RituximabBoltbody (BB-01)] is superior at eliciting CD40 upregulation as comparedto Ritux-ATAC2 and equimolar concentrations of unconjugated rituximab(Roche).

FIG. 67K shows that the BB-01 immunoconjugate produced according to theSATA method [Rituximab Boltbody (BB-01)] is superior at eliciting CD86upregulation on myeloid cells as compared to the rituximab withvaline-citruline-PABC linker immunoconjugate produced according to themethods described in US 2017/0158772 (Ritux-ATAC2) and equimolarconcentrations of unconjugated rituximab (Roche).

FIG. 67L shows CD123 expression on myeloid cells following 18 hours ofstimulation with the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) as compared to unconjugated rituximab(Roche).

FIG. 67M shows CD14 expression on myeloid cells following 18 hours ofstimulation with the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) as compared to unconjugated rituximab(Roche).

FIG. 67N shows CD16 expression on myeloid cells following 18 hours ofstimulation with the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) as compared to unconjugated rituximab(Roche).

FIG. 67O shows CD40 expression on myeloid cells following 18 hours ofstimulation with the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) as compared to unconjugated rituximab(Roche).

FIG. 67P shows CD86 expression on myeloid cells following 18 hours ofstimulation with the rituximab with valine-citruline-PABC linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC2) as compared to unconjugated rituximab(Roche).

FIG. 68A shows that the rituximab immunoconjugate produced according tothe BB-01 SATA method (Rituximab Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to unconjugated rituximab(Roche) as well as equimolar concentrations of comparative conjugatesprepared with rituximab and either a valine-citruline-PABC or amaleimide-PEG4 linker, both containing a pentafluorophenyl group withgardiquimod (Rituximab-ATAC2, Rituximab-ATAC3 respectively; US2017/0158772) following 18 hours of stimulation.

FIG. 68B shows that the rituximab immunoconjugate produced according tothe BB-01 SATA method (Rituximab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to unconjugated rituximab(Roche) as well as equimolar concentrations of comparative conjugatesprepared with rituximab and either a valine-citruline-PABC or amaleimide-PEG4 linker, both containing a pentafluorophenyl group withgardiquimod (Rituximab-ATAC2, Rituximab-ATAC3 respectively; US2017/0158772) following 18 hours of stimulation.

FIG. 68C shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method orthe rituximab immunoconjugates according to the methods described in US2017/0158772 following overnight deglycosylation with PNGase F.

FIG. 68D shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method orthe rituximab immunoconjugates according to the methods described in US2017/0158772.

FIG. 68E shows a liquid chromatography-mass spectrometry analysis of theipsilateral heavy-light chain of the rituximab immunoconjugate producedusing a maleimide-PEG4 linker as described in US 2017/0158772 followingovernight deglycosylation with PNGase F.

FIG. 68F shows a liquid chromatography-mass spectrometry analysis of thelight chain of the rituximab immunoconjugate produced using amaleimide-PEG4 linker as described in US 2017/0158772 followingovernight deglycosylation with PNGase F.

FIG. 68G shows that the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) fails to elicit CD123 upregulation on myeloidcells following 18 hours of stimulation. FIG. 68G also shows that theBB-01 immunoconjugate produced according to the SATA method [RituximabBoltbody (BB-01)] is superior at eliciting CD123 upregulation ascompared to Ritux-ATAC3 and equimolar concentrations of unconjugatedrituximab (Roche).

FIG. 68H shows that the BB-01 immunoconjugate produced according to theSATA method [Rituximab Boltbody (BB-01)] is superior at eliciting CD14downregulation on myeloid cells as compared to the rituximab withmaleimide-PEG4 linker immunoconjugate produced according to the methodsdescribed in US 2017/0158772 (Ritux-ATAC3) and equimolar concentrationsof unconjugated rituximab (Roche) following 18 hours of stimulation.

FIG. 68I shows that the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) fails to elicit CD16 downregulation onmyeloid cells following 18 hours of stimulation. FIG. 68I also showsthat the BB-01 immunoconjugate produced according to the SATA method[Rituximab Boltbody (BB-01)] is superior at eliciting CD40 upregulationas compared to Ritux-ATAC2 and equimolar concentrations of unconjugatedrituximab (Roche).

FIG. 68J shows that the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) fails to elicit CD40 upregulation on myeloidcells following 18 hours of stimulation. FIG. 68J also shows that theBB-01 immunoconjugate produced according to the SATA method [RituximabBoltbody (BB-01)] is superior at eliciting CD40 upregulation as comparedto Ritux-ATAC2 and equimolar concentrations of unconjugated rituximab(Roche).

FIG. 68K shows that the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) fails to elicit CD86 upregulation on myeloidcells following 18 hours of stimulation. FIG. 68J also shows that theBB-01 immunoconjugate produced according to the SATA method [RituximabBoltbody (BB-01)] is superior at eliciting CD86 upregulation as comparedto Ritux-ATAC2 and equimolar concentrations of unconjugated rituximab(Roche).

FIG. 68L shows CD123 expression on myeloid cells following 18 hours ofstimulation with the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) as compared to unconjugated rituximab(Roche).

FIG. 68M shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) as compared to unconjugated rituximab(Roche).

FIG. 68N shows CD14 expression on myeloid cells following 18 hours ofstimulation with the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) as compared to unconjugated rituximab (Roche)

FIG. 68O shows CD16 expression on myeloid cells following 18 hours ofstimulation with the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) as compared to unconjugated rituximab(Roche).

FIG. 68P shows CD40 expression on myeloid cells following 18 hours ofstimulation with the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) as compared to unconjugated rituximab(Roche).

FIG. 68Q shows CD86 expression on myeloid cells following 18 hours ofstimulation with the rituximab with maleimide-PEG4 linkerimmunoconjugate produced according to the methods described in US2017/0158772 (Ritux-ATAC3) as compared to unconjugated rituximab(Roche).

FIG. 69A shows that the atezolizumab immunoconjugate produced accordingto the BB-01 method (Atezolizumab IgG1 NQ Boltbody) elicits superiorIL-10 secretion from myeloid cells as compared to equimolarconcentrations of unconjugated atezolizumab (Roche) following 18 hoursof stimulation.

FIG. 69B shows that the atezolizumab immunoconjugate produced accordingto the BB-01 method (Atezolizumab IgG1 NQ Boltbody) elicits superiorTNFα secretion from myeloid cells as compared to equimolarconcentrations of unconjugated atezolizumab (Roche) following 18 hoursof stimulation.

FIG. 69C shows a liquid chromatography-mass spectrometry analysis of theatezolizumab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 69D shows a liquid chromatography-mass spectrometry analysis ofunconjugated atezolizumab (Roche) that was utilized to produce theatezolizumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 69E shows a liquid chromatography-mass spectrometry analysis ofunconjugated atezolizumab (Roche) that was utilized to produce theatezolizumab immunoconjugate according to the BB-01 method.

FIG. 69F shows CD123 expression on myeloid cells following 18 hours ofstimulation with the atezolizumab immunoconjugate produced according tothe BB-01 method (Atezolizumab Boltbody) as compared to unconjugatedatezolizumab (Roche).

FIG. 69G shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the atezolizumab immunoconjugate produced according tothe BB-01 method (Atezolizumab Boltbody) as compared to unconjugatedatezolizumab (Roche).

FIG. 69H shows that the atezolizumab immunoconjugate produced accordingto the BB-01 method (Atezolizumab Boltbody) is superior at elicitingCD14 downregulation on myeloid cells as compared to the unconjugatedatezolizumab (Roche) following 18 hours of stimulation.

FIG. 69I shows that the atezolizumab immunoconjugate produced accordingto the BB-01 method (Atezolizumab Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to the unconjugatedatezolizumab (Roche) following 18 hours of stimulation.

FIG. 69J shows that the atezolizumab immunoconjugate produced accordingto the BB-01 method (Atezolizumab Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to the unconjugatedatezolizumab (Roche) following 18 hours of stimulation.

FIG. 69K shows CD86 expression on myeloid cells following 18 hours ofstimulation with the atezolizumab immunoconjugate produced according tothe BB-01 method (Atezolizumab Boltbody) as compared to unconjugatedatezolizumab (Roche).

FIG. 70A shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated bevacizumab (Roche) following 18 hours of stimulation.

FIG. 70B shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated bevacizumab (Roche) following 18 hours of stimulation.

FIG. 70C shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated bevacizumab (Roche) following 36 hours of stimulation.

FIG. 70D shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated bevacizumab (Roche) following 36 hours of stimulation.

FIG. 70E shows a liquid chromatography-mass spectrometry analysis of thebevacizumab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 70F shows a liquid chromatography-mass spectrometry analysis ofunconjugated bevacizumab (Roche) that was utilized to produce thebevacizumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 70G shows a liquid chromatography-mass spectrometry analysis ofunconjugated bevacizumab (Roche) that was utilized to produce thebevacizumab immunoconjugate according to the BB-01 method.

FIG. 70H shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to the unconjugatedbevacizumab (Roche) following 18 hours of stimulation.

FIG. 70I shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the bevacizumab immunoconjugate produced according tothe BB-01 method (Bevacizumab Boltbody) as compared to unconjugatedbevacizumab (Roche).

FIG. 70J shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedbevacizumab (Roche) following 18 hours of stimulation.

FIG. 70K shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedbevacizumab (Roche) following 18 hours of stimulation.

FIG. 70L shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugatedbevacizumab (Roche) following 18 hours of stimulation.

FIG. 70M shows that the bevacizumab immunoconjugate produced accordingto the BB-01 method (Bevacizumab Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugatedbevacizumab (Roche) following 18 hours of stimulation.

FIG. 71A shows a liquid chromatography-mass spectrometry analysis of thecetuximab immunoconjugate produced according to the BB-01 conjugationmethod from the cetuximab biosimilar (Alphamab) following overnightdeglycosylation with PNGase F.

FIG. 71B shows a liquid chromatography-mass spectrometry analysis of thecetuximab immunoconjugate produced according to the BB-01 conjugationmethod.

FIG. 71C shows a liquid chromatography-mass spectrometry analysis ofunconjugated cetuximab biosimilar (Alphamab) that was utilized toproduce the cetuximab immunoconjugate according to the BB-01 methodfollowing overnight deglycosylation with PNGase F.

FIG. 71D shows a liquid chromatography-mass spectrometry analysis ofunconjugated cetuximab biosimilar (Alphamab) that was utilized toproduce the cetuximab immunoconjugate according to the BB-01 conjugationmethod.

FIG. 71E shows that the rituximab immunoconjugates produced according tothe BB-01 method from rituximab biosimilars (AmAb, Alphamab; JHL, JHLBiotech; LGM, LGM Pharma) elicit comparable CD123 upregulation onmyeloid cells following 18 hours of stimulation. The dashed lineindicates the level of expression on unstimulated myeloid cells culturedfor 18 hours.

FIG. 71F shows that the rituximab immunoconjugates produced according tothe BB-01 method from rituximab biosimilars (AmAb, Alphamab; JHL, JHLBiotech; LGM, LGM Pharma) elicit comparable HLA-DR expression on myeloidcells following 18 hours of stimulation. The dashed line indicates thelevel of expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 71G shows that the rituximab immunoconjugates produced according tothe BB-01 method from rituximab biosimilars (biosimilar 1, Alphamab;biosimilar 2, JHL Biotech; biosimilar 3, LGM Pharma) elicit comparableCD14 downregulation on myeloid cells following 18 hours of stimulation.The dashed line indicates the level of expression on unstimulatedmyeloid cells cultured for 18 hours.

FIG. 71H shows that the rituximab immunoconjugates produced according tothe BB-01 method from rituximab biosimilars (AmAb, Alphamab; JHL, JHLBiotech; LGM, LGM Pharma) elicit comparable CD16 downregulation onmyeloid cells following 18 hours of stimulation. The dashed lineindicates the level of expression on unstimulated myeloid cells culturedfor 18 hours.

FIG. 71I shows that the rituximab immunoconjugates produced according tothe BB-01 method from rituximab biosimilars (AmAb, Alphamab; JHL, JHLBiotech; LGM, LGM Pharma) elicit comparable CD40 upregulation on myeloidcells following 18 hours of stimulation. The dashed line indicates thelevel of expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 71J shows that the rituximab immunoconjugates produced according tothe BB-01 method from rituximab biosimilars (AmAb, Alphamab; JHL, JHLBiotech; LGM, LGM Pharma) elicit comparable CD86 upregulation on myeloidcells following 18 hours of stimulation. The dashed line indicates thelevel of expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 71K shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (Alphamab) following overnightdeglycosylation with PNGase F.

FIG. 71L shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (Alphamab).

FIG. 71M shows a liquid chromatography-mass spectrometry analysis of anunconjugated rituximab biosimilar (Alphamab) that was utilized toproduce the rituximab biosimilar immunoconjugate according to the BB-01method following overnight deglycosylation with PNGase F.

FIG. 71N shows a liquid chromatography-mass spectrometry analysis of anunconjugated rituximab biosimilar (Alphamab) that was utilized toproduce the rituximab biosimilar immunoconjugate according to the BB-01method.

FIG. 71O shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 1)] is superior at eliciting CD123 upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 1), Alphamab] following 18 hours of stimulation.

FIG. 71P shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 1)] is superior at eliciting HLA-DR upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 1), Alphamab] following 18 hours of stimulation.

FIG. 71Q shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 1)] is superior at eliciting CD14 downregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 1), Alphamab] following 18 hours of stimulation.

FIG. 71R shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 1)] is superior at eliciting CD16 downregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 1), Alphamab] following 18 hours of stimulation.

FIG. 71S shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 1)] is superior at eliciting CD40 upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 1), Alphamab] following 18 hours of stimulation.

FIG. 71T shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 1)] is superior at eliciting CD86 upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 1), Alphamab] following 18 hours of stimulation.

FIG. 71U shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (LGM Pharma) following overnightdeglycosylation with PNGase F.

FIG. 71V shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (LGM Pharma).

FIG. 71W shows a liquid chromatography-mass spectrometry analysis of anunconjugated rituximab biosimilar (LGM Pharma) that was utilized toproduce the rituximab biosimilar immunoconjugate according to the BB-01method following overnight deglycosylation with PNGase F.

FIG. 71X shows a liquid chromatography-mass spectrometry analysis of anunconjugated rituximab biosimilar (LGM Pharma) that was utilized toproduce the rituximab biosimilar immunoconjugate according to the BB-01method.

FIG. 71Y shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar (BB-01) issuperior at eliciting CD123 upregulation on myeloid cells as compared tothe corresponding unconjugated rituximab biosimilar (CD20, LGM Pharma)following 18 hours of stimulation.

FIG. 71Z shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar (BB-01) issuperior at eliciting HLA-DR upregulation on myeloid cells as comparedto the corresponding unconjugated rituximab biosimilar (CD20, LGMPharma) following 18 hours of stimulation.

FIG. 71AA shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar (BB-01) issuperior at eliciting CD14 downregulation on myeloid cells as comparedto the corresponding unconjugated rituximab biosimilar (CD20, LGMPharma) following 18 hours of stimulation.

FIG. 71AB shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar (BB-01) issuperior at eliciting CD16 downregulation on myeloid cells as comparedto the corresponding unconjugated rituximab biosimilar (CD20, LGMPharma) following 18 hours of stimulation.

FIG. 71AC shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar (BB-01) issuperior at eliciting CD40 upregulation on myeloid cells as compared tothe corresponding unconjugated rituximab biosimilar (CD20, LGM Pharma)following 18 hours of stimulation.

FIG. 71AD shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar (BB-01) issuperior at eliciting CD86 upregulation on myeloid cells as compared tothe corresponding unconjugated rituximab biosimilar (CD20, LGM Pharma)following 18 hours of stimulation.

FIG. 71AE shows a liquid chromatography-mass spectrometry analysis ofthe rituximab immunoconjugate produced according to the BB-01conjugation method from the rituximab biosimilar (JHL Biotech) followingovernight deglycosylation with PNGase F.

FIG. 71AF shows a liquid chromatography-mass spectrometry analysis ofthe rituximab immunoconjugate produced according to the BB-01conjugation method from the rituximab biosimilar (JHL Biotech).

FIG. 71AG shows a liquid chromatography-mass spectrometry analysis of anunconjugated rituximab biosimilar (JHL Biotech) that was utilized toproduce the rituximab biosimilar immunoconjugate according to the BB-01method following overnight deglycosylation with PNGase F.

FIG. 71AH shows a liquid chromatography-mass spectrometry analysis of anunconjugated rituximab biosimilar (JHL Biotech) that was utilized toproduce the rituximab biosimilar immunoconjugate according to the BB-01method.

FIG. 71AI shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 2)] is superior at eliciting CD123 upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 2), JHL Biotech] following 18 hours of stimulation.

FIG. 71AJ shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 2)] is superior at eliciting HLA-DR upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 2), JHL Biotech] following 18 hours of stimulation.

FIG. 71AK shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 2)] is superior at eliciting CD40 upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 2), JHL Biotech] following 18 hours of stimulation.

FIG. 71AL shows that the rituximab biosimilar immunoconjugate producedaccording to the BB-01 method from a rituximab biosimilar [BB-01(biosimilar 2)] is superior at eliciting CD86 upregulation on myeloidcells as compared to the corresponding unconjugated rituximab biosimilar[(CD20 (biosimilar 2), JHL Biotech] following 18 hours of stimulation.

FIG. 71AM shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche).

FIG. 71AN shows a liquid chromatography-mass spectrometry analysis ofthe trastuzumab immunoconjugate produced according to the BB-01conjugation method from the trastuzumab biosimilar (JHL Biotech)following overnight deglycosylation with PNGase F.

FIG. 71AO shows a liquid chromatography-mass spectrometry analysis ofthe trastuzumab immunoconjugate produced according to the BB-01conjugation method from the trastuzumab biosimilar (JHL Biotech).

FIG. 71AP shows a liquid chromatography-mass spectrometry analysis of anunconjugated trastuzumab biosimilar (JHL Biotech) that was utilized toproduce the trastuzumab biosimilar immunoconjugate according to theBB-01 method following overnight deglycosylation with PNGase F.

FIG. 71AQ shows a liquid chromatography-mass spectrometry analysis of anunconjugated trastuzumab biosimilar (JHL Biotech) that was utilized toproduce the trastuzumab biosimilar immunoconjugate according to theBB-01 method.

FIG. 71AR shows that the trastuzumab biosimilar immunoconjugate producedaccording to the BB-01 method from a trastuzumab biosimilar (BB-40) issuperior at eliciting CD123 upregulation on myeloid cells as compared tothe corresponding unconjugated trastuzumab biosimilar [Trastuzumab(JHL), JHL Biotech) following 18 hours of stimulation.

FIG. 71AS shows that the trastuzumab biosimilar immunoconjugate producedaccording to the BB-01 method from a trastuzumab biosimilar (BB-40) issuperior at eliciting HLA-DR upregulation on myeloid cells as comparedto the corresponding unconjugated trastuzumab biosimilar [Trastuzumab(JHL), JHL Biotech) following 18 hours of stimulation.

FIG. 71AT shows that the trastuzumab biosimilar immunoconjugate producedaccording to the BB-01 method from a trastuzumab biosimilar (BB-40) issuperior at eliciting CD14 downregulation on myeloid cells as comparedto the corresponding unconjugated trastuzumab biosimilar [Trastuzumab(JHL), JHL Biotech) following 18 hours of stimulation.

FIG. 71AU shows that the trastuzumab biosimilar immunoconjugate producedaccording to the BB-01 method from a trastuzumab biosimilar (BB-40) issuperior at eliciting CD16 downregulation on myeloid cells as comparedto the corresponding unconjugated trastuzumab biosimilar [Trastuzumab(JHL), JHL Biotech) following 18 hours of stimulation.

FIG. 71AV shows that the trastuzumab biosimilar immunoconjugate producedaccording to the BB-01 method from a trastuzumab biosimilar (BB-40) issuperior at eliciting CD40 upregulation on myeloid cells as compared tothe corresponding unconjugated trastuzumab biosimilar [Trastuzumab(JHL), JHL Biotech) following 18 hours of stimulation.

FIG. 71AW shows that the trastuzumab biosimilar immunoconjugate producedaccording to the BB-01 method from a trastuzumab biosimilar (BB-40) issuperior at eliciting CD86 upregulation on myeloid cells as compared tothe corresponding unconjugated trastuzumab biosimilar [Trastuzumab(JHL), JHL Biotech) following 18 hours of stimulation.

FIG. 72A shows that the cetuximab immunoconjugate produced according tothe BB-01 method (Cetuximab Boltbody) elicits superior IL-1β secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated cetuximab (Imclone/Lilly) following 18 hours ofstimulation.

FIG. 72B shows that the cetuximab immunoconjugate produced according tothe BB-01 method (Cetuximab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated cetuximab (Imclone/Lilly) following 18 hours ofstimulation.

FIG. 72C shows a liquid chromatography-mass spectrometry analysis ofunconjugated cetuximab (Imclone/Lilly) that was utilized to produce thecetuximab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 72D shows a liquid chromatography-mass spectrometry analysis ofunconjugated cetuximab (Imclone/Lilly) that was utilized to produce thecetuximab immunoconjugate according to the BB-01 conjugation method.

FIG. 72E shows that the cetuximab immunoconjugate produced according tothe BB-01 method (closed circles, red) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated cetuximab(closed squares, black; Imclone/Lilly) following 18 hours ofstimulation.

FIG. 72F shows that the cetuximab immunoconjugate produced according tothe BB-01 method (closed circles, red) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated cetuximab(closed squares, black; Imclone/Lilly) following 18 hours ofstimulation.

FIG. 72G shows that the cetuximab immunoconjugate produced according tothe BB-01 method (closed circles, red) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedcetuximab (closed squares, black; Imclone/Lilly) following 18 hours ofstimulation.

FIG. 72H shows that the cetuximab immunoconjugate produced according tothe BB-01 method (closed circles, red) is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedcetuximab (closed squares, black; Imclone/Lilly) following 18 hours ofstimulation.

FIG. 72I shows that the cetuximab immunoconjugate produced according tothe BB-01 method (closed circles, red) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated cetuximab(closed squares, black; Imclone/Lilly) following 18 hours ofstimulation.

FIG. 72J shows that the cetuximab immunoconjugate produced according tothe BB-01 method (closed circles, red) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated cetuximab(closed squares, black; Imclone/Lilly) following 18 hours ofstimulation.

FIG. 73A shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratumumab Boltbody) elicits superior IL-11secretion from myeloid cells as compared to equimolar concentrations ofunconjugated daratumumab (Genmab/Janssen Biotech) following 18 hours ofstimulation.

FIG. 73B shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratumumab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated daratumumab (Genmab/Janssen Biotech) following 18 hours ofstimulation.

FIG. 73C shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratuzumab [sic] Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated daratumumab (Daratuzumab [sic], Genmab/Janssen Biotech)following 36 hours of stimulation.

FIG. 73D shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratuzumab [sic] Boltbody) elicits superior IL-11secretion from myeloid cells as compared to equimolar concentrations ofunconjugated daratumumab (Daratuzumab [sic], Genmab/Janssen Biotech)following 36 hours of stimulation.

FIG. 73E shows a liquid chromatography-mass spectrometry analysis ofunconjugated daratumumab (Genmab/Janssen Biotech) that was utilized toproduce the daratumumab immunoconjugate according to the BB-01 methodfollowing overnight deglycosylation with PNGase F.

FIG. 73F shows a liquid chromatography-mass spectrometry analysis of thedaratumumab immunoconjugate produced according to the BB-01 methodfollowing overnight deglycosylation with PNGase F.

FIG. 73G shows a liquid chromatography-mass spectrometry analysis ofunconjugated daratumumab (Genmab/Janssen Biotech) that was utilized toproduce the daratumumab immunoconjugate according to the BB-01 method.

FIG. 73H shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratuzumab [sic] Boltbody) is superior ateliciting CD123 upregulation on myeloid cells as compared to theunconjugated daratumumab (Daratuzumab [sic], Genmab/Janssen Biotech)following 18 hours of stimulation.

FIG. 73I shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the daratumumab immunoconjugate produced according tothe BB-01 method (Daratumumab Boltbody) as compared to unconjugateddaratumumab (Genmab/Janssen Biotech).

FIG. 73J shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratuzumab [sic] Boltbody) is superior ateliciting CD14 downregulation on myeloid cells as compared to theunconjugated daratumumab (Daratuzumab [sic], Genmab/Janssen Biotech)following 18 hours of stimulation.

FIG. 73K shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratuzumab [sic] Boltbody) is superior ateliciting CD16 downregulation on myeloid cells as compared to theunconjugated daratumumab (Daratuzumab [sic], Genmab/Janssen Biotech)following 18 hours of stimulation.

FIG. 73L shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratuzumab [sic] Boltbody) is superior ateliciting CD40 upregulation on myeloid cells as compared to theunconjugated daratumumab (Daratuzumab [sic], Genmab/Janssen Biotech)following 18 hours of stimulation.

FIG. 73M shows that the daratumumab immunoconjugate produced accordingto the BB-01 method (Daratuzumab [sic] Boltbody) is superior ateliciting CD86 upregulation on myeloid cells as compared to theunconjugated daratumumab (Daratuzumab [sic], Genmab/Janssen Biotech)following 18 hours of stimulation.

FIG. 74A shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) elicits superior IL-1β secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated elotuzumab (BMS) following 36 hours of stimulation.

FIG. 74B shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated elotuzumab (BMS) following 36 hours of stimulation.

FIG. 74C shows a liquid chromatography-mass spectrometry analysis of theelotuzumab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 74D shows a liquid chromatography-mass spectrometry analysis ofunconjugated elotuzumab (BMS) that was utilized to produce theelotuzumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 74E shows a liquid chromatography-mass spectrometry analysis ofunconjugated elotuzumab (BMS) that was utilized to produce theelotuzumab immunoconjugate according to the BB-01 method.

FIG. 74F shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated elotuzumab(BMS) following 18 hours of stimulation.

FIG. 74G shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated elotuzumab(BMS) following 18 hours of stimulation.

FIG. 74H shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedelotuzumab (BMS) following 18 hours of stimulation.

FIG. 74I shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedelotuzumab (BMS) following 18 hours of stimulation.

FIG. 74J shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated elotuzumab(BMS) following 18 hours of stimulation.

FIG. 74K shows that the elotuzumab immunoconjugate produced according tothe BB-01 method (Elotuzumab Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated elotuzumab(BMS) following 18 hours of stimulation.

FIG. 75A shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated ipilimumab (BMS) following 36 hours of stimulation.

FIG. 75B shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) elicits superior IL-1β secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated ipilimumab (BMS) following 18 hours of stimulation.

FIG. 75C shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated ipilimumab (BMS) following 18 hours of stimulation.

FIG. 75D shows a liquid chromatography-mass spectrometry analysis ofunconjugated ipilimumab (BMS) that was utilized to produce theipilimumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 75E shows a liquid chromatography-mass spectrometry analysis ofunconjugated ipilimumab (BMS) that was utilized to produce theipilimumab immunoconjugate according to the BB-01 method.

FIG. 75F shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated ipilimumab(BMS) following 18 hours of stimulation.

FIG. 75G shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated ipilimumab(BMS) following 18 hours of stimulation.

FIG. 75H shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedipilimumab (BMS) following 18 hours of stimulation.

FIG. 75I shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedipilimumab (BMS) following 18 hours of stimulation.

FIG. 75J shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated ipilimumab(BMS) following 18 hours of stimulation.

FIG. 75K shows that the ipilimumab immunoconjugate produced according tothe BB-01 method (Ipilimumab Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated ipilimumab(BMS) following 18 hours of stimulation.

FIG. 76A shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab IgG4 Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated nivolumab (Nivolumab IgG4, BMS) following 18 hours ofstimulation.

FIG. 76B shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab IgG4 Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated nivolumab (Nivolumab IgG4, BMS) following 18 hours ofstimulation.

FIG. 76C shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab IgG4 Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated nivolumab (Nivolumab IgG4, BMS) following 36 hours ofstimulation.

FIG. 76D shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab IgG4 Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated nivolumab (Nivolumab IgG4, BMS) following 36 hours ofstimulation.

FIG. 76E shows a liquid chromatography-mass spectrometry analysis of thenivolumab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 76F shows a liquid chromatography-mass spectrometry analysis ofunconjugated nivolumab (BMS) that was utilized to produce the nivolumabimmunoconjugate according to the BB-01 conjugation method followingovernight deglycosylation with PNGase F.

FIG. 76G shows a liquid chromatography-mass spectrometry analysis ofunconjugated nivolumab (BMS) that was utilized to produce the nivolumabimmunoconjugate according to the BB-01 method.

FIG. 76H shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab Boltbody) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated nivolumab(BMS) following 18 hours of stimulation.

FIG. 76I shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the nivolumab immunoconjugate produced according to theBB-01 method (Nivolumab Boltbody) as compared to unconjugated nivolumab(BMS).

FIG. 76J shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatednivolumab (BMS) following 18 hours of stimulation.

FIG. 76K shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab Boltbody) is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatednivolumab (BMS) following 18 hours of stimulation.

FIG. 76L shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated nivolumab(BMS) following 18 hours of stimulation.

FIG. 76M shows that the nivolumab immunoconjugate produced according tothe BB-01 method (Nivolumab Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated nivolumab(BMS) following 18 hours of stimulation.

FIG. 77A shows that the obinutuzumab immunoconjugate produced accordingto the BB-01 method (Obinutuzumab Boltbody) elicits superior IL-10secretion from myeloid cells as compared to equimolar concentrations ofunconjugated obinutuzumab (Roche) following 36 hours of stimulation.

FIG. 77B shows that the obinutuzumab immunoconjugate produced accordingto the BB-01 method (Obinutuzumab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated obinutuzumab (Roche) following 36 hours of stimulation.

FIG. 77C shows a liquid chromatography-mass spectrometry analysis ofunconjugated obinutuzumab (Roche) that was utilized to produce theobinutuzumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 77D shows a liquid chromatography-mass spectrometry analysis ofunconjugated obinutuzumab (Roche) that was utilized to produce theobinutuzumab immunoconjugate according to the BB-01 conjugation method.

FIG. 77E shows that the obinutuzumab immunoconjugate produced accordingto the BB-01 method (Obinutuzumab Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to the unconjugated CD20mAb (Roche) following 18 hours of stimulation.

FIG. 77F shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the obinutuzumab immunoconjugate produced according tothe BB-01 method (Obinutuzumab Boltbody) as compared to unconjugatedCD20 mAb (Roche).

FIG. 77G shows that the obinutuzumab immunoconjugate produced accordingto the BB-01 method (Obinutuzumab Boltbody) is superior at elicitingCD14 downregulation on myeloid cells as compared to the unconjugatedCD20 mAb (Roche) following 18 hours of stimulation.

FIG. 77H shows that the obinutuzumab immunoconjugate produced accordingto the BB-01 method (Obinutuzumab Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to the unconjugatedCD20 mAb (Roche) following 18 hours of stimulation.

FIG. 77I shows that the obinutuzumab immunoconjugate produced accordingto the BB-01 method (Obinutuzumab Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to the unconjugated CD20mAb (Roche) following 18 hours of stimulation.

FIG. 77J shows that the obinutuzumab immunoconjugate produced accordingto the BB-01 method (Obinutuzumab Boltbody) is superior at elicitingCD86 upregulation on myeloid cells as compared to the unconjugated CD20mAb (Roche) following 18 hours of stimulation.

FIG. 78A shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) elicits superior IL-1β secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated olaratumab (Lilly) following 36 hours of stimulation.

FIG. 78B shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated olaratumab (Lilly) following 36 hours of stimulation.

FIG. 78C shows a liquid chromatography-mass spectrometry analysis of theolaratumab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 78D shows a liquid chromatography-mass spectrometry analysis ofunconjugated olaratumab (Lilly) that was utilized to produce theolaratumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 78E shows a liquid chromatography-mass spectrometry analysis ofunconjugated olaratumab (Lilly) that was utilized to produce theolaratumab immunoconjugate according to the BB-01 conjugation method.

FIG. 78F shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated olaratumab(Lilly) following 18 hours of stimulation.

FIG. 78G shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated olaratumab(Lilly) following 18 hours of stimulation.

FIG. 78H shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedolaratumab (Lilly) following 18 hours of stimulation.

FIG. 78I shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedolaratumab (Lilly) following 18 hours of stimulation.

FIG. 78J shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated olaratumab(Lilly) following 18 hours of stimulation.

FIG. 78K shows that the olaratumab immunoconjugate produced according tothe BB-01 method (Olaratumab Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated olaratumab(Lilly) following 18 hours of stimulation.

FIG. 79A shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated pembrolizumab (Merck) following 36 hours of stimulation.

FIG. 79B shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated pembrolizumab (Merck) following 36 hours of stimulation.

FIG. 79C shows a liquid chromatography-mass spectrometry analysis of thepembrolizumab immunoconjugate produced according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 79D shows a liquid chromatography-mass spectrometry analysis ofunconjugated pembrolizumab (Merck) that was utilized to produce thepembrolizumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 79E shows a liquid chromatography-mass spectrometry analysis ofunconjugated pembrolizumab (Merck) that was utilized to produce thepembrolizumab immunoconjugate according to the BB-01 conjugation method.

FIG. 79F shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to the unconjugatedpembrolizumab (Merck) following 18 hours of stimulation.

FIG. 79G shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) is superior at elicitingHLA-DR upregulation on myeloid cells as compared to the unconjugatedpembrolizumab (Merck) following 18 hours of stimulation.

FIG. 79H shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) is superior at elicitingCD14 downregulation on myeloid cells as compared to the unconjugatedpembrolizumab (Merck) following 18 hours of stimulation.

FIG. 79I shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to the unconjugatedpembrolizumab (Merck) following 18 hours of stimulation.

FIG. 79J shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to the unconjugatedpembrolizumab (Merck) following 18 hours of stimulation.

FIG. 79K shows that the pembrolizumab immunoconjugate produced accordingto the BB-01 method (Pembrolizumab Boltbody) is superior at elicitingCD86 upregulation on myeloid cells as compared to the unconjugatedpembrolizumab (Merck) following 18 hours of stimulation.

FIG. 80A shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) elicits superior IL-10 secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated pertuzumab (Roche) following 18 hours of stimulation.

FIG. 80B shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated pertuzumab (Roche) following 18 hours of stimulation.

FIG. 80C shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) elicits superior IL-10 secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated pertuzumab (Roche) following 36 hours of stimulation.

FIG. 80D shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated pertuzumab (Roche) following 36 hours of stimulation.

FIG. 80E shows a liquid chromatography-mass spectrometry analysis of thepertuzumab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 80F shows a liquid chromatography-mass spectrometry analysis ofunconjugated pertuzumab (Roche) that was utilized to produce thepertuzumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 80G shows a liquid chromatography-mass spectrometry analysis ofunconjugated pertuzumab (Roche) that was utilized to produce thepertuzumab immunoconjugate according to the BB-01 conjugation method.

FIG. 80H shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated pertuzumab(Roche) following 18 hours of stimulation.

FIG. 80I shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated pertuzumab(Roche) following 18 hours of stimulation.

FIG. 80J shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedpertuzumab (Roche) following 18 hours of stimulation.

FIG. 80K shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedpertuzumab (Roche) following 18 hours of stimulation.

FIG. 80L shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated pertuzumab(Roche) following 18 hours of stimulation.

FIG. 80M shows that the pertuzumab immunoconjugate produced according tothe BB-01 method (Pertuzumab Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated pertuzumab(Roche) following 18 hours of stimulation.

FIG. 81A shows that the rituximab immunoconjugate produced according tothe BB-01 method (Rituximab Boltbody) elicits superior IL-1β secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated rituximab (Roche) following 36 hours of stimulation.

FIG. 81B shows that the rituximab immunoconjugate produced according tothe BB-01 method (Rituximab Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated rituximab (Roche) following 36 hours of stimulation.

FIG. 81C shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 81D shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 81E shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod.

FIG. 81F shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 81G shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method.

FIG. 81H shows that the rituximab immunoconjugate produced according tothe BB-01 method (BB-01) is superior at eliciting CD123 upregulation onmyeloid cells as compared to the unconjugated rituximab (CD20, Roche)following 18 hours of stimulation.

FIG. 81I shows that the rituximab immunoconjugate produced according tothe BB-01 method (BB-01) is superior at eliciting HLA-DR upregulation onmyeloid cells as compared to the unconjugated rituximab (CD20, Roche)following 18 hours of stimulation.

FIG. 81J shows that the rituximab immunoconjugate produced according tothe BB-01 method (BB-01) is superior at eliciting CD14 downregulation onmyeloid cells as compared to the unconjugated rituximab (CD20, Roche)following 18 hours of stimulation.

FIG. 81K shows that the rituximab immunoconjugate produced according tothe BB-01 method (BB-01) is superior at eliciting CD16 downregulation onmyeloid cells as compared to the unconjugated rituximab (CD20, Roche)following 18 hours of stimulation.

FIG. 81L shows that the rituximab immunoconjugate produced according tothe BB-01 method (BB-01) is superior at eliciting CD40 upregulation onmyeloid cells as compared to the unconjugated rituximab (CD20, Roche)following 18 hours of stimulation.

FIG. 81M shows that the rituximab immunoconjugate produced according tothe BB-01 method (BB-01) is superior at eliciting CD86 upregulation onmyeloid cells as compared to the unconjugated rituximab (CD20, Roche)following 18 hours of stimulation.

FIG. 82A shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (Trastuzumab Boltbody) elicits superior IL-1βsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated trastuzumab (Roche) following 36 hours of stimulation.

FIG. 82B shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (Trastuzumab Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations ofunconjugated trastuzumab (Roche) following 36 hours of stimulation.

FIG. 82C shows a liquid chromatography-mass spectrometry analysis of thetrastuzumab immunoconjugate produced according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 82D shows a liquid chromatography-mass spectrometry analysis ofunconjugated trastuzumab (Roche) that was utilized to produce thetrastuzumab immunoconjugate according to the BB-01 conjugation methodfollowing overnight deglycosylation with PNGase F.

FIG. 82E shows a liquid chromatography-mass spectrometry analysis ofunconjugated trastuzumab (Roche) that was utilized to produce thetrastuzumab immunoconjugate according to the BB-01 conjugation method.

FIG. 82F shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (closed circles, red) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugatedtrastuzumab (closed squares, black; Roche) following 18 hours ofstimulation.

FIG. 82G shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (closed circles, red) is superior at elicitingHLA-DR upregulation on myeloid cells as compared to the unconjugatedtrastuzumab (closed squares, black; Roche) following 18 hours ofstimulation.

FIG. 82H shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (closed circles, red) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedtrastuzumab (closed squares, black; Roche) following 18 hours ofstimulation.

FIG. 82I shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (closed circles, red is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedtrastuzumab (closed squares, black; Roche) following 18 hours ofstimulation.

FIG. 82J shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (closed circles, red) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugatedtrastuzumab (closed squares, black; Roche) following 18 hours ofstimulation.

FIG. 82K shows that the trastuzumab immunoconjugate produced accordingto the BB-01 method (closed circles, red) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugatedtrastuzumab (closed squares, black; Roche) following 18 hours ofstimulation.

FIG. 83A shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody) elicits superior IL-10 secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated etanercept (Amgen) following 36 hours of stimulation.

FIG. 83B shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody) elicits superior TNFα secretionfrom myeloid cells as compared to equimolar concentrations ofunconjugated etanercept (Amgen) following 36 hours of stimulation.

FIG. 83C shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the unconjugatedetanercept (Amgen) following 18 hours of stimulation.

FIG. 83D shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody is superior at eliciting CD16downregulation on myeloid cells as compared to the unconjugatedetanercept (Amgen) following 18 hours of stimulation.

FIG. 83E shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated etanercept(Amgen) following 18 hours of stimulation.

FIG. 83F shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated etanercept(Amgen) following 18 hours of stimulation.

FIG. 83G shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated etanercept(Amgen) following 18 hours of stimulation.

FIG. 83H shows that the etanercept immunoconjugate produced according tothe BB-01 method (Etanercept Boltbody) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated etanercept(Amgen) following 18 hours of stimulation.

FIG. 84A shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (LGM Pharma). The calculated DAR is0.7.

FIG. 84B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab biosimilar (LGM Pharma) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 84C shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (LGM Pharma) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method.

FIG. 84D shows CD14 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 0.7)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 84E shows CD16 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 0.7)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 84F shows CD40 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 0.7)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 84G shows CD86 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 0.7)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 84H shows CD123 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 0.7)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 84I shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 0.7)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 85A shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (LGM Pharma). The calculated DAR is1.6.

FIG. 85B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab biosimilar (LGM Pharma) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 85C shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (LGM Pharma) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method.

FIG. 85D shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (LGM Pharma) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method.

FIG. 85E shows CD14 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 85F shows CD16 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 85G shows CD40 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 85H shows CD86 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 85I shows CD123 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 85J shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 86A shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (LGM Pharma). The calculated DAR is2.5.

FIG. 86B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab biosimilar (LGM Pharma) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 86C shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (LGM Pharma) that was utilized to produce therituximab immunoconjugate according to the BB-01 conjugation method.

FIG. 86D shows a liquid chromatography-mass spectrometry analysis of therituximab immunoconjugate produced according to the BB-01 conjugationmethod from the rituximab biosimilar (LGM Pharma). The calculated DAR is2.5.

FIG. 86E shows CD14 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 2.5)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 86F shows CD16 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 2.5)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 86G shows CD40 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 2.5)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 86H shows CD86 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 2.5)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 86I shows CD123 expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 86J shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the rituximab immunoconjugate produced according to theBB-01 method [BB-01 (DAR 1.6)]. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 87A shows that the rituximab immunoconjugates of varying DAR, allproduced according to the BB-01 method from the rituximab biosimilar(LGM Pharma) elicit comparable CD14 downregulation on myeloid cellsfollowing 18 hours of stimulation. The dashed line indicates the levelof expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 87B shows that the rituximab immunoconjugates of varying DAR, allproduced according to the BB-01 method from the rituximab biosimilar(LGM Pharma) elicit comparable CD16 downregulation on myeloid cellsfollowing 18 hours of stimulation. The dashed line indicates the levelof expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 87C shows that the rituximab immunoconjugates of varying DAR, allproduced according to the BB-01 method from the rituximab biosimilar(LGM Pharma) elicit comparable CD40 upregulation on myeloid cellsfollowing 18 hours of stimulation. The dashed line indicates the levelof expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 87D shows that the rituximab immunoconjugates of varying DAR, allproduced according to the BB-01 method from the rituximab biosimilar(LGM Pharma) elicit comparable CD86 upregulation on myeloid cellsfollowing 18 hours of stimulation. The dashed line indicates the levelof expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 87E shows that the rituximab immunoconjugates of varying DAR, allproduced according to the BB-01 method from the rituximab biosimilar(LGM Pharma) elicit comparable CD123 upregulation on myeloid cellsfollowing 18 hours of stimulation. The dashed line indicates the levelof expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 87F shows that the rituximab immunoconjugates of varying DAR, allproduced according to the BB-01 method from the rituximab biosimilar(LGM Pharma) elicit comparable HLA-DR upregulation on myeloid cellsfollowing 18 hours of stimulation. The dashed line indicates the levelof expression on unstimulated myeloid cells cultured for 18 hours.

FIG. 88A shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab IgA2 (Invivogen, hcd20-mab7) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 88B shows a liquid chromatography-mass spectrometry analysis of therituximab IgA2 immunoconjugate produced according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 88C shows that the rituximab IgA2 immunoconjugate producedaccording to the BB-01 method (CD20 IgA2 Boltbody) is superior ateliciting CD14 downregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgA2; Invivogen, hcd20-mac7) following 18hours of stimulation.

FIG. 88D shows that the rituximab IgA2 immunoconjugate producedaccording to the BB-01 method (CD20 IgA2 Boltbody) is superior ateliciting CD16 downregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgA2; Invivogen, hcd20-mac7) following 18hours of stimulation.

FIG. 88E shows that the rituximab IgA2 immunoconjugate producedaccording to the BB-01 method (CD20 IgA2 Boltbody) is superior ateliciting CD40 upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgA2; Invivogen, hcd20-mac7) following 18hours of stimulation.

FIG. 88F shows that the rituximab IgA2 immunoconjugate producedaccording to the BB-01 method (CD20 IgA2 Boltbody) is superior ateliciting CD86 upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgA2; Invivogen, hcd20-mac7) following 18hours of stimulation.

FIG. 88G shows that the rituximab IgA2 immunoconjugate producedaccording to the BB-01 method (CD20 IgA2 Boltbody) is superior ateliciting CD123 upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgA2; Invivogen, hcd20-mac7) following 18hours of stimulation.

FIG. 88H shows that the rituximab IgA2 immunoconjugate producedaccording to the BB-01 method (CD20 IgA2 Boltbody) is superior ateliciting HLA-DR upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgA2; Invivogen, hcd20-mac7) following 18hours of stimulation.

FIG. 89A shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (IgG1 Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations(0.2 μM) of unconjugated rituximab (IgG; Invivogen, hcd20-mab1)following 36 hours of stimulation.

FIG. 89B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab IgG1 (Invivogen, hcd20-mab1) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod.

FIG. 89C shows a liquid chromatography-mass spectrometry analysis of therituximab IgG1 immunoconjugate produced according to the BB-01conjugation method.

FIG. 89D shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (CD20 IgG1 Boltbody) is superior ateliciting CD14 downregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgG1; Invivogen, hcd20-mab1) following 18hours of stimulation.

FIG. 89E shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (CD20 IgG1 Boltbody) is superior ateliciting CD16 downregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgG1; Invivogen, hcd20-mab1) following 18hours of stimulation.

FIG. 89F shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (CD20 IgG1 Boltbody) is superior ateliciting CD40 upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgG1; Invivogen, hcd20-mab1) following 18hours of stimulation.

FIG. 89G shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (CD20 IgG1 Boltbody) is superior ateliciting CD86 upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgG1; Invivogen, hcd20-mab1) following 18hours of stimulation.

FIG. 89H shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (CD20 IgG1 Boltbody) is superior ateliciting CD123 upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgG; Invivogen, hcd20-mab1) following 18hours of stimulation.

FIG. 89I shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (CD20 IgG1 Boltbody) is superior ateliciting HLA-DR upregulation on myeloid cells as compared to theunconjugated rituximab (CD20 IgG; Invivogen, hcd20-mab1) following 18hours of stimulation.

FIG. 90A shows that the rituximab afucosylated IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 AF Boltbody) elicitssuperior TNFα secretion from myeloid cells as compared to equimolarconcentrations (0.2 μM) of unconjugated rituximab (IgG1 AF; Invivogen,hcd20-mab13) following 18 hours of stimulation.

FIG. 90B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab IgG1 (Invivogen, hcd20-mab13) that was utilizedto produce the rituximab immunoconjugate according to the BB-01conjugation method.

FIG. 90C shows a liquid chromatography-mass spectrometry analysis of therituximab IgG1 immunoconjugate produced according to the BB-01conjugation method.

FIG. 90D shows that the rituximab IgG1 AF immunoconjugate producedaccording to the BB-01 method (IgG1 AF Boltbody) is superior ateliciting CD14 downregulation on myeloid cells as compared to theunconjugated rituximab (IgG1 AF; Invivogen, hcd20-mab13) following 18hours of stimulation.

FIG. 90E shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (IgG1 AF Boltbody) is superior ateliciting CD16 downregulation on myeloid cells as compared to theunconjugated rituximab (IgG1 AF; Invivogen, hcd20-mab13) following 18hours of stimulation.

FIG. 90F shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (IgG1 AF Boltbody) is superior ateliciting CD40 upregulation on myeloid cells as compared to theunconjugated rituximab (IgG1 AF; Invivogen, hcd20-mab13) following 18hours of stimulation.

FIG. 90G shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (IgG1 AF Boltbody) is superior ateliciting CD86 upregulation on myeloid cells as compared to theunconjugated rituximab (IgG1 AF; Invivogen, hcd20-mab13) following 18hours of stimulation.

FIG. 90H shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (IgG1 AF Boltbody) is superior ateliciting CD123 upregulation on myeloid cells as compared to theunconjugated rituximab (IgG1 AF; Invivogen, hcd20-mab13) following 18hours of stimulation.

FIG. 90I shows that the rituximab IgG1 immunoconjugate producedaccording to the BB-01 method (IgG1 AF Boltbody) is superior ateliciting HLA-DR upregulation on myeloid cells as compared to theunconjugated rituximab (IgG1 AF; Invivogen, hcd20-mab13) following 18hours of stimulation.

FIG. 91A shows that the rituximab N297Q mutant IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 NQ Boltbody) elicitssuperior TNFαsecretion from myeloid cells as compared to equimolarconcentrations (0. μM) of unconjugated rituximab (IgG1 NQ; Invivogen,hcd20-mab12) following 36 hours of stimulation.

FIG. 91B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab IgG1 (Invivogen, hcd20-mab12) that was utilizedto produce the rituximab immunoconjugate according to the BB-01conjugation method.

FIG. 91C shows a liquid chromatography-mass spectrometry analysis of therituximab IgG1 immunoconjugate produced according to the BB-01conjugation method.

FIG. 91D shows CD14 expression on myeloid cells following 18 hours ofstimulation with the rituximab N297Q mutant IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 NQ Boltbody) as compared tounconjugated rituximab IgG1 (Invivogen, hcd20-mab12).

FIG. 91E shows CD16 expression on myeloid cells following 18 hours ofstimulation with the rituximab N297Q mutant IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 NQ Boltbody) as compared tounconjugated rituximab IgG1 (Invivogen, hcd20-mab12).

FIG. 91F shows CD40 expression on myeloid cells following 18 hours ofstimulation with the rituximab N297Q mutant IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 NQ Boltbody) as compared tounconjugated rituximab IgG1 (Invivogen, hcd20-mab12).

FIG. 91G shows CD86 expression on myeloid cells following 18 hours ofstimulation with the rituximab N297Q mutant IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 NQ Boltbody) as compared tounconjugated rituximab IgG1 (Invivogen, hcd20-mab12).

FIG. 91H shows CD123 expression on myeloid cells following 18 hours ofstimulation with the rituximab N297Q mutant IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 NQ Boltbody) as compared tounconjugated rituximab IgG1 (Invivogen, hcd20-mab12).

FIG. 91I shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the rituximab N297Q mutant IgG1 immunoconjugateproduced according to the BB-01 method (IgG1 NQ Boltbody) as compared tounconjugated rituximab IgG1 (Invivogen, hcd20-mab12).

FIG. 92A shows that the rituximab IgG2 immunoconjugate producedaccording to the BB-01 method (IgG2 Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations(0.2 μM) of unconjugated rituximab (IgG2; Invivogen, hcd20-mab2)following 18 hours of stimulation.

FIG. 92B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab IgG2 (Invivogen, hcd20-mab2) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod.

FIG. 92C shows a liquid chromatography-mass spectrometry analysis of therituximab IgG2 immunoconjugate produced according to the BB-01conjugation method.

FIG. 92D shows that the rituximab IgG2 immunoconjugate producedaccording to the BB-01 method (IgG2 Boltbody) is superior at elicitingCD14 downregulation on myeloid cells as compared to the unconjugatedrituximab (IgG2; Invivogen, hcd20-mab2) following 18 hours ofstimulation.

FIG. 92E shows that the rituximab IgG2 immunoconjugate producedaccording to the BB-01 method (IgG2 Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to the unconjugatedrituximab (IgG2; Invivogen, hcd20-mab2) following 18 hours ofstimulation.

FIG. 92F shows that the rituximab IgG2 immunoconjugate producedaccording to the BB-01 method (IgG2 Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG2; Invivogen, hcd20-mab2) following 18 hours ofstimulation.

FIG. 92G shows that the rituximab IgG2 immunoconjugate producedaccording to the BB-01 method (IgG2 Boltbody) is superior at elicitingCD86 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG2; Invivogen, hcd20-mab2) following 18 hours ofstimulation.

FIG. 92H shows that the rituximab IgG2 immunoconjugate producedaccording to the BB-01 method (IgG2 Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG2; Invivogen, hcd20-mab2) following 18 hours ofstimulation.

FIG. 92I shows that the rituximab IgG2 immunoconjugate producedaccording to the BB-01 method (IgG2 Boltbody) is superior at elicitingHLA-DR upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG2; Invivogen, hcd20-mab2) following 18 hours ofstimulation.

FIG. 93A shows that the rituximab IgG3 immunoconjugate producedaccording to the BB-01 method (IgG3 Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations(0.2 μM) of unconjugated rituximab (IgG3; Invivogen, hcd20-mab3)following 18 hours of stimulation.

FIG. 93B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab IgG3 (Invivogen, hcd20-mab3) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod.

FIG. 93C shows a liquid chromatography-mass spectrometry analysis of therituximab IgG3 immunoconjugate produced according to the BB-01conjugation method.

FIG. 93D shows that the rituximab IgG3 immunoconjugate producedaccording to the BB-01 method (IgG3 Boltbody) is superior at elicitingCD14 downregulation on myeloid cells as compared to the unconjugatedrituximab (IgG3; Invivogen, hcd20-mab3) following 18 hours ofstimulation.

FIG. 93E shows that the rituximab IgG3 immunoconjugate producedaccording to the BB-01 method (IgG3 Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to the unconjugatedrituximab (IgG3; Invivogen, hcd20-mab3) following 18 hours ofstimulation.

FIG. 93F shows that the rituximab IgG3 immunoconjugate producedaccording to the BB-01 method (IgG3 Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG3; Invivogen, hcd20-mab3) following 18 hours ofstimulation.

FIG. 93G shows that the rituximab IgG3 immunoconjugate producedaccording to the BB-01 method (IgG3 Boltbody) is superior at elicitingCD86 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG3; Invivogen, hcd20-mab3) following 18 hours ofstimulation.

FIG. 93H shows that the rituximab IgG3 immunoconjugate producedaccording to the BB-01 method (IgG3 Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG3; Invivogen, hcd20-mab3) following 18 hours ofstimulation.

FIG. 93I shows that the rituximab IgG3 immunoconjugate producedaccording to the BB-01 method (IgG3 Boltbody) is superior at elicitingHLA-DR upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG3; Invivogen, hcd20-mab3) following 18 hours ofstimulation.

FIG. 94A shows that the rituximab IgG4 immunoconjugate producedaccording to the BB-01 method (IgG4 Boltbody) elicits superior TNFαsecretion from myeloid cells as compared to equimolar concentrations(0.2 μM) of unconjugated rituximab (IgG4; Invivogen, hcd20-mab4)following 18 hours of stimulation.

FIG. 94B shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab IgG4 (Invivogen, hcd20-mab4) that was utilized toproduce the rituximab immunoconjugate according to the BB-01 conjugationmethod.

FIG. 94C shows a liquid chromatography-mass spectrometry analysis of therituximab IgG4 immunoconjugate produced according to the BB-01conjugation method.

FIG. 94D shows that the rituximab IgG4 immunoconjugate producedaccording to the BB-01 method (IgG4 Boltbody) is superior at elicitingCD14 downregulation on myeloid cells as compared to the unconjugatedrituximab (IgG4; Invivogen, hcd20-mab4) following 18 hours ofstimulation.

FIG. 94E shows that the rituximab IgG4 immunoconjugate producedaccording to the BB-01 method (IgG4 Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to the unconjugatedrituximab (IgG4; Invivogen, hcd20-mab4) following 18 hours ofstimulation.

FIG. 94F shows that the rituximab IgG4 immunoconjugate producedaccording to the BB-01 method (IgG4 Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG4; Invivogen, hcd20-mab4) following 18 hours ofstimulation.

FIG. 94G shows that the rituximab IgG4 immunoconjugate producedaccording to the BB-01 method (IgG4 Boltbody) is superior at elicitingCD86 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG4; Invivogen, hcd20-mab4) following 18 hours ofstimulation.

FIG. 94H shows that the rituximab IgG4 immunoconjugate producedaccording to the BB-01 method (IgG4 Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG4; Invivogen, hcd20-mab4) following 18 hours ofstimulation.

FIG. 94I shows that the rituximab IgG4 immunoconjugate producedaccording to the BB-01 method (IgG4 Boltbody) is superior at elicitingHLA-DR upregulation on myeloid cells as compared to the unconjugatedrituximab (IgG4; Invivogen, hcd20-mab4) following 18 hours ofstimulation.

FIG. 95 is a table that lists the EC50 values and fold-changes of CD14,CD40, and CD86 expression for IgG1 Boltbody (BB-IgG1), IgG1 AF Boltbody(BB-IgG1 AF), IgG2 Boltbody (BB-IgG2), IgG3 Boltbody (BB-IgG3), and IgG4Boltbody (BB-IgG4) referenced in FIGS. 89, 90, 92, 93, and 94respectively. EC50 values were computed based on dose-response curvesgenerated from 5-fold serial dilutions. All fold-changes were calculatedrelative to the respective naked antibody at the indicatedconcentration.

FIG. 96A shows a liquid chromatography-mass spectrometry analysis ofunconjugated atezolizumab IgG1 isotype variant (Invivogen, hpd11-mab1)that was utilized to produce the atezolizumab immunoconjugate accordingto the BB-01 conjugation method.

FIG. 96B shows a liquid chromatography-mass spectrometry analysis of theatezolizumab IgG1 isotype variant immunoconjugate produced according tothe BB-01 conjugation method.

FIG. 96C shows CD14 expression on myeloid cells following 18 hours ofstimulation with the atezolizumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Atezolizumab—IgG1 Boltbody) ascompared to unconjugated atezolizumab (Atezolizumab—IgG; Invivogen,hpd11-mab1).

FIG. 96D shows CD16 expression on myeloid cells following 18 hours ofstimulation with the atezolizumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Atezolizumab—IgG1 Boltbody) ascompared to unconjugated atezolizumab (Atezolizumab—IgG; Invivogen,hpd11-mab1).

FIG. 96E shows CD40 expression on myeloid cells following 18 hours ofstimulation with the atezolizumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Atezolizumab—IgG1 Boltbody) ascompared to unconjugated atezolizumab (Atezolizumab—IgG1; Invivogen,hpd11-mab1).

FIG. 96F shows CD86 expression on myeloid cells following 18 hours ofstimulation with the atezolizumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Atezolizumab—IgG1 Boltbody) ascompared to unconjugated atezolizumab (Atezolizumab—IgG; Invivogen,hpd11-mab1).

FIG. 96G shows CD123 expression on myeloid cells following 18 hours ofstimulation with the atezolizumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Atezolizumab—IgG1 Boltbody) ascompared to unconjugated atezolizumab (Atezolizumab—IgG1; Invivogen,hpd11-mab1).

FIG. 96H shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the atezolizumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Atezolizumab—IgG1 Boltbody) ascompared to unconjugated atezolizumab (Atezolizumab—IgG1; Invivogen,hpd11-mab1).

FIG. 97A shows a liquid chromatography-mass spectrometry analysis ofunconjugated nivolumab IgG1 isotype variant (Invivogen, hpd1ni-mab1)that was utilized to produce the nivolumab immunoconjugate according tothe BB-01 conjugation method.

FIG. 97B shows a liquid chromatography-mass spectrometry analysis of thenivolumab IgG1 isotype variant immunoconjugate produced according to theBB-01 conjugation method.

FIG. 97C shows CD14 expression on myeloid cells following 18 hours ofstimulation with the nivolumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Nivolumab—IgG1 Boltbody) ascompared to unconjugated nivolumab (Nivolumab—IgG; Invivogen,hpd1ni-mab1).

FIG. 97D shows CD16 expression on myeloid cells following 18 hours ofstimulation with the nivolumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Nivolumab—IgG1 Boltbody) ascompared to unconjugated nivolumab (Nivolumab—IgG1; Invivogen,hpd1ni-mab1).

FIG. 97E shows CD40 expression on myeloid cells following 18 hours ofstimulation with the nivolumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Nivolumab—IgG1 Boltbody) ascompared to unconjugated nivolumab (Nivolumab—IgG1; Invivogen,hpd1ni-mab1).

FIG. 97F shows CD86 expression on myeloid cells following 18 hours ofstimulation with the nivolumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Nivolumab—IgG1 Boltbody) ascompared to unconjugated nivolumab (Nivolumab—IgG1; Invivogen,hpd1ni-mab1).

FIG. 97G shows CD123 expression on myeloid cells following 18 hours ofstimulation with the nivolumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Nivolumab—IgG1 Boltbody) ascompared to unconjugated nivolumab (Nivolumab—IgG1; Invivogen,hpd1ni-mab1).

FIG. 97H shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the nivolumab IgG1 isotype variant immunoconjugateproduced according to the BB-01 method (Nivolumab—IgG1 Boltbody) ascompared to unconjugated nivolumab (Nivolumab—IgG1; Invivogen,hpd1ni-mab1).

FIG. 98A shows a liquid chromatography-mass spectrometry analysis ofunconjugated anti-gp75 mAb (BioXcell, TA99-BE0151) that was utilized toproduce the anti-gp75 mAb immunoconjugate according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 98B shows a liquid chromatography-mass spectrometry analysis of theanti-gp75 mAb immunoconjugate produced according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 98C shows CD14 expression on myeloid cells following 18 hours ofstimulation with the anti-gp75 mAb immunoconjugate produced according tothe BB-01 method (GP75 Boltbody.

FIG. 98D shows CD16 expression on myeloid cells following 18 hours ofstimulation with the anti-gp75 mAb immunoconjugate produced according tothe BB-01 method (GP75 Boltbody).

FIG. 98E shows CD40 expression on myeloid cells following 18 hours ofstimulation with the anti-gp75 mAb immunoconjugate produced according tothe BB-01 method (GP75 Boltbody.

FIG. 98F shows CD86 expression on myeloid cells following 18 hours ofstimulation with the anti-gp75 mAb immunoconjugate produced according tothe BB-01 method (GP75 Boltbody).

FIG. 98G shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the anti-gp75 mAb immunoconjugate produced according tothe BB-01 method (GP75 Boltbody).

FIG. 99A shows a liquid chromatography-mass spectrometry analysis of theunconjugated rituximab biosimilar (CD20, LGM Pharma) that was utilizedto produce the rituximab immunoconjugate according to the BB-03conjugation method following overnight deglycosylation with PNGase F.

FIG. 99B shows a liquid chromatography-mass spectrometry analysis of theunconjugated rituximab biosimilar (CD20, LGM Pharma) that was utilizedto produce the rituximab immunoconjugate according to the BB-03conjugation method.

FIG. 99C shows a liquid chromatography-mass spectrometry analysis of theBB-03 immunoconjugate produced according to the BB-03 conjugationmethod.

FIG. 99D shows that the BB-03 immunoconjugate produced according to theBB-03 method (BB-03) is superior at eliciting CD123 upregulation onmyeloid cells as compared to the unconjugated rituximab biosimilar(CD20; LGM Pharma) following 18 hours of stimulation.

FIG. 99E shows that the BB-03 immunoconjugate produced according to theBB-03 method (BB-03) is superior at eliciting HLA-DR upregulation onmyeloid cells as compared to the unconjugated rituximab biosimilar(CD20; LGM Pharma) following 18 hours of stimulation.

FIG. 99F shows that the BB-03 immunoconjugate produced according to theBB-03 method (BB-03) is superior at eliciting CD14 downregulation onmyeloid cells as compared to the unconjugated rituximab biosimilar(CD20; LGM Pharma) following 18 hours of stimulation.

FIG. 99G shows that the BB-03 immunoconjugate produced according to theBB-03 method (BB-03) is superior at eliciting CD16 downregulation onmyeloid cells as compared to the unconjugated rituximab biosimilar(CD20; LGM Pharma) following 18 hours of stimulation.

FIG. 99H shows that the BB-03 immunoconjugate produced according to theBB-03 method (BB-03) is superior at eliciting CD40 upregulation onmyeloid cells as compared to the unconjugated rituximab biosimilar(CD20; LGM Pharma) following 18 hours of stimulation.

FIG. 99I shows that the BB-03 immunoconjugate produced according to theBB-03 method (BB-03) is superior at eliciting CD86 upregulation onmyeloid cells as compared to the unconjugated rituximab biosimilar(CD20; LGM Pharma) following 18 hours of stimulation.

FIG. 100A shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-05conjugation method following overnight deglycosylation with PNGase F.

FIG. 100B shows a liquid chromatography-mass spectrometry analysis ofthe BB-05 immunoconjugate produced according to the BB-05 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 100C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-05conjugation method.

FIG. 100D shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-05 immunoconjugate produced according to theBB-05 method (BB-05). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 100E shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-05 immunoconjugate produced according to theBB-05 method (BB-05). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 100F shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-05 immunoconjugate produced according to theBB-05 method (BB-05). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 100G shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-05 immunoconjugate produced according to theBB-05 method (BB-05). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 100H shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-05 immunoconjugate produced according to theBB-05 method (BB-05). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 100I shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-05 immunoconjugate produced according to theBB-05 method (BB-05). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 101A shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-06conjugation method following overnight deglycosylation with PNGase F.

FIG. 101B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-06conjugation method.

FIG. 101C shows a liquid chromatography-mass spectrometry analysis ofthe BB-06 immunoconjugate produced according to the BB-06 conjugationmethod.

FIG. 102A shows a liquid chromatography-mass spectrometry analysis ofthe BB-07 immunoconjugate produced according to the BB-07 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 102B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-07conjugation method following overnight deglycosylation with PNGase F.

FIG. 102C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-07conjugation method.

FIG. 102D shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours. FIG. 102D alsocompares BB-07 to the BB-01 immunoconjugate produced according to theBB-01 conjugation method (BB-01).

FIG. 102E shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours. FIG. 102E alsocompares BB-07 to the BB-01 immunoconjugate produced according to theBB-01 conjugation method (BB-01).

FIG. 102F shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours. FIG. 102F alsocompares BB-07 to the BB-01 immunoconjugate produced according to theBB-01 conjugation method (BB-01).

FIG. 102G shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours. FIG. 102G alsocompares BB-07 to the BB-01 immunoconjugate produced according to theBB-01 conjugation method (BB-01).

FIG. 102H shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours. FIG. 102H alsocompares BB-07 to the BB-01 immunoconjugate produced according to theBB-01 conjugation method (BB-01).

FIG. 102I shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours. FIG. 102I alsocompares BB-07 to the BB-01 immunoconjugate produced according to theBB-01 conjugation method (BB-01).

FIG. 102J shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 102K shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 102L shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 102M shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours. FIG. 102M alsocompares BB-07 to the BB-01 immunoconjugate produced according to theBB-01 conjugation method (BB-01).

FIG. 102N shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-07 immunoconjugate produced according to theBB-07 method (BB-07). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 103A shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-11conjugation method following overnight deglycosylation with PNGase F.

FIG. 103B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-11conjugation method.

FIG. 103C shows a liquid chromatography-mass spectrometry analysis ofthe BB-11 immunoconjugate produced according to the BB-11 conjugationmethod following overnight deglycosylation with PNGase F.

FIG. 103D shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-11 immunoconjugate produced according to theBB-11 method (BB-11). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 103E shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-11 immunoconjugate produced according to theBB-11 method (BB-11). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 103F shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-11 immunoconjugate produced according to theBB-11 method (BB-11). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 103G shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-11 immunoconjugate produced according to theBB-11 method (BB-11). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 103H shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-11 immunoconjugate produced according to theBB-11 method (BB-11). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 103I shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-11 immunoconjugate produced according to theBB-11 method (BB-11). The dashed line indicates the level of expressionon unstimulated myeloid cells cultured for 18 hours.

FIG. 104A shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-14PFP conjugation method.

FIG. 104B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-14PFP conjugation method following overnight deglycosylation with PNGaseF.

FIG. 104C shows a liquid chromatography-mass spectrometry analysis ofthe BB-14 immunoconjugate produced according to the BB-14 PFPconjugation method.

FIG. 104D shows that the rituximab immunoconjugate produced according tothe BB-14 PFP conjugation method (BB-14) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 104E shows that the rituximab immunoconjugate produced according tothe BB-14 PFP conjugation method (BB-14) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 104F shows that the rituximab immunoconjugate produced according tothe BB-14 PFP conjugation method (BB-14) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 104G shows that the rituximab immunoconjugate produced according tothe BB-14 PFP conjugation method (BB-14) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 104H shows that the rituximab immunoconjugate produced according tothe BB-14 PFP conjugation method (BB-14) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 104I shows that the rituximab immunoconjugate produced according tothe BB-14 PFP conjugation method (BB-14) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 105A shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-15NHS conjugation method following overnight deglycosylation with PNGaseF.

FIG. 105B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-15NHS conjugation method.

FIG. 105C shows a liquid chromatography-mass spectrometry analysis ofthe BB-15 immunoconjugate produced according to the BB-15 NHSconjugation method.

FIG. 105D shows that the rituximab immunoconjugate produced according tothe BB-15 NHS conjugation method (BB-15) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 105E shows that the rituximab immunoconjugate produced according tothe BB-15 NHS conjugation method (BB-15) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 105F shows that the rituximab immunoconjugate produced according tothe BB-15 NHS conjugation method (BB-15) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 105G shows that the rituximab immunoconjugate produced according tothe BB-15 NHS conjugation method (BB-15) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 105H shows that the rituximab immunoconjugate produced according tothe BB-15 NHS conjugation method (BB-15) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 105I shows that the rituximab immunoconjugate produced according tothe BB-15 NHS conjugation method (BB-15) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 106A shows a liquid chromatography-mass spectrometry analysis ofthe BB-17 immunoconjugate produced according to the BB-17 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 106B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-17TFP conjugation method following overnight deglycosylation with PNGaseF.

FIG. 106C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-17TFP conjugation method.

FIG. 106D shows that the rituximab immunoconjugate produced according tothe BB-17 TFP conjugation method (BB-17) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 106E shows that the rituximab immunoconjugate produced according tothe BB-17 TFP conjugation method (BB-17) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 106F shows that the rituximab immunoconjugate produced according tothe BB-17 TFP conjugation method (BB-17) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 106G shows that the rituximab immunoconjugate produced according tothe BB-17 TFP conjugation method (BB-17) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 106H shows that the rituximab immunoconjugate produced according tothe BB-17 TFP conjugation method (BB-17) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 106I shows that the rituximab immunoconjugate produced according tothe BB-17 TFP conjugation method (BB-17) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 107A shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-22SATA conjugation method following overnight deglycosylation with PNGaseF.

FIG. 107B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-22SATA conjugation method.

FIG. 107C shows a liquid chromatography-mass spectrometry analysis ofthe BB-22 immunoconjugate produced according to the BB-22 SATAconjugation method.

FIG. 108A shows a liquid chromatography-mass spectrometry analysis ofthe BB-24 immunoconjugate produced according to the BB-24 TFPconjugation method.

FIG. 108B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-24 TFPconjugation method.

FIG. 108C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-24 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 108D shows that the rituximab immunoconjugate produced according tothe BB-24 TFP conjugation method (BB-24) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 108E shows that the rituximab immunoconjugate produced according tothe BB-24 TFP conjugation method (BB-24) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 108F shows that the rituximab immunoconjugate produced according tothe BB-24 TFP conjugation method (BB-24) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 108G shows that the rituximab immunoconjugate produced according tothe BB-24 TFP conjugation method (BB-24) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 108H shows that the rituximab immunoconjugate produced according tothe BB-24 TFP conjugation method (BB-24) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 108I shows that the rituximab immunoconjugate produced according tothe BB-24 TFP conjugation method (BB-24) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 109A shows a liquid chromatography-mass spectrometry analysis ofthe BB-26 immunoconjugate produced according to the BB-26 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 109B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-26TFP conjugation method following overnight deglycosylation with PNGaseF.

FIG. 109C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-26TFP conjugation method.

FIG. 109D shows that the rituximab immunoconjugate produced according tothe BB-26 TFP conjugation method (BB-26) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 109E shows that the rituximab immunoconjugate produced according tothe BB-26 TFP conjugation method (BB-26) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 109F shows that the rituximab immunoconjugate produced according tothe BB-26 TFP conjugation method (BB-26) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 109G shows that the rituximab immunoconjugate produced according tothe BB-26 TFP conjugation method (BB-26) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 110A shows that the rituximab immunoconjugate produced according tothe BB-27 TFP conjugation method (BB-27) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 110B shows that the rituximab immunoconjugate produced according tothe BB-27 TFP conjugation method (BB-27) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 110C shows that the rituximab immunoconjugate produced according tothe BB-27 TFP conjugation method (BB-27) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 110D shows that the rituximab immunoconjugate produced according tothe BB-27 TFP conjugation method (BB-27) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 110E shows that the rituximab immunoconjugate produced according tothe BB-27 TFP conjugation method (BB-27) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 110F shows that the rituximab immunoconjugate produced according tothe BB-27 TFP conjugation method (BB-27) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, LGM Pharma) following 18 hours of stimulation.

FIG. 110G shows a liquid chromatography-mass spectrometry analysis ofthe BB-27 immunoconjugate produced according to the BB-27 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 110H shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-27TFP conjugation method following overnight deglycosylation with PNGaseF.

FIG. 110I shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, LGM Pharma) that wasutilized to produce the rituximab immunoconjugate according to the BB-27TFP conjugation method.

FIG. 111A shows a liquid chromatography-mass spectrometry analysis ofthe BB-36 immunoconjugate produced according to the BB-36 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 111B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-36 TFPconjugation method.

FIG. 111C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-36 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 111D shows that the rituximab immunoconjugate produced according tothe BB-36 TFP conjugation method (BB-36) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 111E shows that the rituximab immunoconjugate produced according tothe BB-36 TFP conjugation method (BB-36) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 111F shows that the rituximab immunoconjugate produced according tothe BB-36 TFP conjugation method (BB-36) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 111G shows that the rituximab immunoconjugate produced according tothe BB-36 TFP conjugation method (BB-36) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 111H shows that the rituximab immunoconjugate produced according tothe BB-36 TFP conjugation method (BB-36) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 111I shows that the rituximab immunoconjugate produced according tothe BB-36 TFP conjugation method (BB-36) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 112A shows a liquid chromatography-mass spectrometry analysis ofthe BB-37 immunoconjugate produced according to the BB-37 TFPconjugation method.

FIG. 112B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-37 TFPconjugation method.

FIG. 112C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-37 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 112D shows that the rituximab immunoconjugate produced according tothe BB-37 TFP conjugation method (BB-37) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 112E shows that the rituximab immunoconjugate produced according tothe BB-37 TFP conjugation method (BB-37) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 112F shows that the rituximab immunoconjugate produced according tothe BB-37 TFP conjugation method (BB-37) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 112G shows that the rituximab immunoconjugate produced according tothe BB-37 TFP conjugation method (BB-37) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 112H shows that the rituximab immunoconjugate produced according tothe BB-37 TFP conjugation method (BB-37) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 112I shows that the rituximab immunoconjugate produced according tothe BB-37 TFP conjugation method (BB-37) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 113A shows a liquid chromatography-mass spectrometry analysis ofthe BB-45 immunoconjugate produced according to the BB-45 TFPconjugation method.

FIG. 113B shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-45 TFPconjugation method.

FIG. 113C shows a liquid chromatography-mass spectrometry analysis ofthe unconjugated rituximab biosimilar (CD20, Alphamab) that was utilizedto produce the rituximab immunoconjugate according to the BB-45 TFPconjugation method following overnight deglycosylation with PNGase F.

FIG. 113D shows that the rituximab immunoconjugate produced according tothe BB-45 TFP conjugation method (BB-45) is superior at eliciting CD123upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 113E shows that the rituximab immunoconjugate produced according tothe BB-45 TFP conjugation method (BB-45) is superior at eliciting HLA-DRupregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 113F shows that the rituximab immunoconjugate produced according tothe BB-45 TFP conjugation method (BB-45) is superior at eliciting CD14upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 113G shows that the rituximab immunoconjugate produced according tothe BB-45 TFP conjugation method (BB-45) is superior at eliciting CD16upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 113H shows that the rituximab immunoconjugate produced according tothe BB-45 TFP conjugation method (BB-45) is superior at eliciting CD40upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 113I shows that the rituximab immunoconjugate produced according tothe BB-45 TFP conjugation method (BB-45) is superior at eliciting CD86upregulation on myeloid cells as compared to the unconjugated rituximab(CD20, Alphamab) following 18 hours of stimulation.

FIG. 114A shows a liquid chromatography-mass spectrometry analysis ofthe heavy chain of an unconjugated CD40 monoclonal antibody (Bioxcell,BE0016-2)

FIG. 114B shows a liquid chromatography-mass spectrometry analysis ofthe light chain of an unconjugated CD40 monoclonal antibody (Bioxcell,BE0016-2).

FIG. 114C shows a liquid chromatography-mass spectrometry analysis ofthe heavy chain of a CD40 immunoconjugate produced according to US2017/0158772.

FIG. 114D shows a liquid chromatography-mass spectrometry analysis ofthe light chain of a CD40 immunoconjugate produced according to US2017/0158772.

FIG. 115A shows a liquid chromatography-mass spectrometry analysis of anunconjugated CD40 monoclonal antibody (Bioxcell, BE0016-2).

FIG. 115B shows a liquid chromatography-mass spectrometry analysis of aCD40 immunoconjugate produced according to the BB-01 conjugation method.

FIG. 116A shows a liquid chromatography-mass spectrometry analysis of anunconjugated CLEC5a monoclonal antibody (R&D Systems, mab1639).

FIG. 116B shows a liquid chromatography-mass spectrometry analysis of aCLEC5a immunoconjugate produced according to the BB-01 conjugationmethod.

FIG. 117A shows a schematic for a CL264 immunoconjugate producedaccording to BB-01 conjugation method.

FIG. 117B shows a schematic for a CL264 immunoconjugate producedaccording to the ester synthesis method.

FIG. 118A shows that the Rituximab-CL307 immunoconjugate producedaccording to the BB-01 conjugation method upregulates CD123 on myeloidcells in a dose-dependent manner following 18 hours of stimulation. Thedashed line indicates the level of expression on unstimulated cellscultured for 18 hours.

FIG. 118B shows that the Rituximab-CL307 immunoconjugate producedaccording to the BB-01 conjugation method upregulates HLA-DR on myeloidcells in a dose-dependent manner following 18 hours of stimulation. Thedashed line indicates the level of expression on unstimulated cellscultured for 18 hours.

FIG. 118C shows that the Rituximab-CL307 immunoconjugate producedaccording to the BB-01 conjugation method downregulates CD14 on myeloidcells in a dose-dependent manner following 18 hours of stimulation. Thedashed line indicates the level of expression on unstimulated cellscultured for 18 hours.

FIG. 118D shows that the Rituximab-CL307 immunoconjugate producedaccording to the BB-01 conjugation method downregulates CD16 on myeloidcells in a dose-dependent manner following 18 hours of stimulation. Thedashed line indicates the level of expression on unstimulated cellscultured for 18 hours.

FIG. 118E shows that the Rituximab-CL307 immunoconjugate producedaccording to the BB-01 conjugation method upregulates CD40 on myeloidcells in a dose-dependent manner following 18 hours of stimulation. Thedashed line indicates the level of expression on unstimulated cellscultured for 18 hours.

FIG. 118F shows that the Rituximab-CL307 immunoconjugate producedaccording to the BB-01 conjugation method upregulates CD86 on myeloidcells in a dose-dependent manner following 18 hours of stimulation. Thedashed line indicates the level of expression on unstimulated cellscultured for 18 hours.

FIG. 118G shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-CL307.

FIG. 118H shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-CL307 following overnight deglycosylation with PNGase F.

FIG. 118I shows that the Rituximab-CL307 immunoconjugate producedaccording to the BB-01 conjugation method elicits TNFα secretion in adose-dependent manner following 18 hours of stimulation.

FIG. 118J shows a liquid chromatography-mass spectrometry analysis ofthe Rituximab-CL307 immunoconjugate produced according to the BB-01conjugation method.

FIG. 119A shows that the Rituximab-CL419 immunoconjugate producedaccording to the BB-01 method (Rituximab-CL419 Boltbody) is superior ateliciting IL-1β secretion from myeloid cells as compared to unconjugatedRituximab (Roche) following 36 hours of stimulation.

FIG. 119B shows that the Rituximab-CL419 immunoconjugate producedaccording to the BB-01 method (Rituximab-CL419 Boltbody) is superior ateliciting TNFα secretion from myeloid cells as compared to unconjugatedRituximab (Roche) following 36 hours of stimulation.

FIG. 119C shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-CL419 following overnight deglycosylation with PNGase F.

FIG. 119D shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-CL419.

FIG. 119E shows a liquid chromatography-mass spectrometry analysis ofthe Rituximab-CL419 immunoconjugate produced according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 119F shows that the Rituximab-CL419 immunoconjugate producedaccording to the BB-01 method (CL419 Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 119G shows that the Rituximab-CL419 immunoconjugate producedaccording to the BB-01 method (CL419 Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to unconjugated Rituximab(CD20; Roche) following 18 hours of stimulation.

FIG. 119H shows that the Rituximab-CL419 immunoconjugate producedaccording to the BB-01 method (CL419 Boltbody) is superior at elicitingCD86 upregulation on myeloid cells as compared to unconjugated Rituximab(CD20; Roche) following 18 hours of stimulation.

FIG. 119I shows that the Rituximab-CL419 immunoconjugate producedaccording to the BB-01 method (CL419 Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 120A shows that the Rituximab-CL572 immunoconjugate producedaccording to the BB-01 method (Rituximab-CL572 Boltbody) is superior ateliciting IL-1β secretion from myeloid cells as compared to unconjugatedRituximab (Roche) following 36 hours of stimulation.

FIG. 120B shows that the Rituximab-CL572 immunoconjugate producedaccording to the BB-01 method (Rituximab-CL572 Boltbody) is superior ateliciting TNFα secretion from myeloid cells as compared to unconjugatedRituximab (Roche) following 36 hours of stimulation.

FIG. 120C shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-CL572 following overnight deglycosylation with PNGase F.

FIG. 120D shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-CL572.

FIG. 120E shows a liquid chromatography-mass spectrometry analysis ofthe Rituximab-CL572 immunoconjugate produced according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 120F shows that the Rituximab-CL572 immunoconjugate producedaccording to the BB-01 method (CL572 Boltbody) is superior at elicitingCD123 upregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 120G shows that the Rituximab-CL572 immunoconjugate producedaccording to the BB-01 method (CL572 Boltbody) is superior at elicitingHLA-DR upregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 120H shows that the Rituximab-CL572 immunoconjugate producedaccording to the BB-01 method (CL572 Boltbody) is superior at elicitingCD16 downregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 120I shows that the Rituximab-CL572 immunoconjugate producedaccording to the BB-01 method (CL572 Boltbody) is superior at elicitingCD40 upregulation on myeloid cells as compared to unconjugated Rituximab(CD20; Roche) following 18 hours of stimulation.

FIG. 121A shows that the Rituximab-Pam2CSK4 immunoconjugate producedaccording to the BB-01 method (Rituximab-Pam2CSK4 Boltbody) is superiorat eliciting IL-10 secretion from myeloid cells as compared tounconjugated Rituximab (Roche) following 36 hours of stimulation.

FIG. 121B shows that the Rituximab-Pam2CSK4 immunoconjugate producedaccording to the BB-01 method (Rituximab-Pam2CSK4 Boltbody) is superiorat eliciting TNFα secretion from myeloid cells as compared tounconjugated Rituximab (Roche) following 36 hours of stimulation.

FIG. 121C shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-Pam2CSK4 following overnight deglycosylation with PNGase F.

FIG. 121D shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-Pam2CSK4.

FIG. 121E shows a liquid chromatography-mass spectrometry analysis ofthe Rituximab-Pam2CSK4 immunoconjugate produced according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 121F shows that the Rituximab-Pam2CSK4 immunoconjugate producedaccording to the BB-01 method (Pam2CSK4 Boltbody) is superior ateliciting CD16 downregulation on myeloid cells as compared tounconjugated Rituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 121G shows that the Rituximab-Pam2CSK4 immunoconjugate producedaccording to the BB-01 method (Pam2CSK4 Boltbody) is superior ateliciting CD40 upregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 121H shows that the Rituximab-Pam2CSK4 immunoconjugate producedaccording to the BB-01 method (Pam2CSK4 Boltbody) is superior ateliciting CD86 upregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 122A shows that the Rituximab-Pam3CSK4 immunoconjugate producedaccording to the BB-01 method (Rituximab-Pam3CSK4 Boltbody) is superiorat eliciting IL-10 secretion from myeloid cells as compared tounconjugated Rituximab (Roche) following 36 hours of stimulation.

FIG. 122B shows that the Rituximab-Pam3CSK4 immunoconjugate producedaccording to the BB-01 method (Rituximab-Pam3CSK4 Boltbody) is superiorat eliciting TNFα secretion from myeloid cells as compared tounconjugated Rituximab (Roche) following 36 hours of stimulation.

FIG. 122C shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-Pam3CSK4 following overnight deglycosylation with PNGase F.

FIG. 122D shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-Pam3CSK4.

FIG. 122E shows a liquid chromatography-mass spectrometry analysis ofthe Rituximab-Pam3CSK4 immunoconjugate produced according to the BB-01conjugation method following overnight deglycosylation with PNGase F.

FIG. 122F shows that the Rituximab-Pam3CSK4 immunoconjugate producedaccording to the BB-01 method (Pam3CSK4 Boltbody) is superior ateliciting CD16 downregulation on myeloid cells as compared tounconjugated Rituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 122G shows that the Rituximab-Pam3CSK4 immunoconjugate producedaccording to the BB-01 method (Pam3CSK4 Boltbody) is superior ateliciting CD40 upregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 122H shows that the Rituximab-Pam3CSK4 immunoconjugate producedaccording to the BB-01 method (Pam3CSK4 Boltbody) is superior ateliciting CD86 upregulation on myeloid cells as compared to unconjugatedRituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 122I shows that the Rituximab-Pam3CSK4 immunoconjugate producedaccording to the BB-01 method (Pam3CSK4 Boltbody) is superior ateliciting CD123 upregulation on myeloid cells as compared tounconjugated Rituximab (CD20; Roche) following 18 hours of stimulation.

FIG. 123A shows a liquid chromatography-mass spectrometry analysis ofthe BB-43 immunoconjugate produced according to the TFP conjugationmethod.

FIG. 123B shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-43 following overnight deglycosylation with PNGase F.

FIG. 123C shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-43.

FIG. 123D shows that the BB-43 immunoconjugate produced according to theTFP method is superior at eliciting CD123 upregulation on myeloid cellsas compared to an unconjugated Rituximab biosimilar (LGM Pharma)following 18 hours of stimulation.

FIG. 123E shows that the BB-43 immunoconjugate produced according to theTFP method is superior at eliciting HLA-DR upregulation on myeloid cellsas compared to an unconjugated Rituximab biosimilar (LGM Pharma)following 18 hours of stimulation.

FIG. 123F shows that the BB-43 immunoconjugate produced according to theTFP method is superior at eliciting CD14 downregulation on myeloid cellsas compared to an unconjugated Rituximab biosimilar (LGM Pharma)following 18 hours of stimulation.

FIG. 123G shows that the BB-43 immunoconjugate produced according to theTFP method is superior at eliciting CD16 downregulation on myeloid cellsas compared to an unconjugated Rituximab biosimilar (LGM Pharma)following 18 hours of stimulation.

FIG. 123H shows that the BB-43 immunoconjugate produced according to theTFP method is superior at eliciting CD40 upregulation on myeloid cellsas compared to an unconjugated Rituximab biosimilar (LGM Pharma)following 18 hours of stimulation.

FIG. 123I shows that the BB-43 immunoconjugate produced according to theTFP method is superior at eliciting CD86 upregulation on myeloid cellsas compared to an unconjugated Rituximab biosimilar (LGM Pharma)following 18 hours of stimulation.

FIG. 124A shows that the Rituximab-SATA-T782 immunoconjugate producedaccording to the BB-01 method (Rituximab-SATA-T782 Boltbody) elicitsTNFα secretion from myeloid cells in a dose-dependent manner following18 hours of stimulation.

FIG. 124B shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-SATA-T782 following overnight deglycosylation with PNGase F.

FIG. 124C shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-SATA-T782.

FIG. 124D shows a liquid chromatography-mass spectrometry analysis ofRituximab-SATA-T782 produced according to the BB-01 method.

FIG. 124E shows that the Rituximab-SATA-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATA-T782)upregulates CD123 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 124F shows that the Rituximab-SATA-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATA-T782)upregulates HLA-DR on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 124G shows that the Rituximab-SATA-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATA-T782)downregulates CD14 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 124H shows that the Rituximab-SATA-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATA-T782)downregulates CD16 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 124I shows that the Rituximab-SATA-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATA-T782)upregulates CD40 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 124J shows that the Rituximab-SATA-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATA-T782)upregulates CD86 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 125A shows that the Rituximab-SATP-T782 immunoconjugate producedaccording to the BB-01 method (Rituximab-SATP-T782 Boltbody) elicitsTNFα secretion from myeloid cells in a dose-dependent manner following18 hours of stimulation.

FIG. 125B shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-SATP-T782 following overnight deglycosylation with PNGase F.

FIG. 125C shows a liquid chromatography-mass spectrometry analysis ofunconjugated Rituximab (Roche) that was utilized to produceRituximab-SATP-T782.

FIG. 125D shows a liquid chromatography-mass spectrometry analysis ofRituximab-SATP-T782 produced according to the BB-01 method.

FIG. 125E shows that the Rituximab-SATP-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATP-T782)upregulates CD123 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 125F shows that the Rituximab-SATP-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATP-T782)upregulates HLA-DR on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 125G shows that the Rituximab-SATP-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATP-T782)downregulates CD14 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 125H shows that the Rituximab-SATP-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATP-T782)downregulates CD16 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 125I shows that the Rituximab-SATP-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATP-T782)upregulates CD40 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 125J shows that the Rituximab-SATP-T782 immunoconjugate producedaccording to the BB-01 conjugation method (Rituximab-SATP-T782)upregulates CD86 on myeloid cells in a dose-dependent manner following18 hours of stimulation. The dashed line indicates the level ofexpression on unstimulated myeloid cells cultured for 18 hours.

FIG. 126A shows a liquid chromatography-mass spectrometry analysis ofthe BB-12 immunoconjugate produced according to the SATA conjugationmethod following overnight conjugation with PNGase F.

FIG. 126B shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-12 following overnight deglycosylation with PNGase F.

FIG. 126C shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-12.

FIG. 126D shows that the BB-12 immunoconjugate produced according to theSATA method fails to elicit CD123 upregulation following 18 hours ofstimulation. FIG. 126D also shows that the BB-11 immunoconjugateproduced according to the SATA method is superior at eliciting CD123upregulation as compared to BB-12 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-11 and BB-12 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 126E shows that the BB-12 immunoconjugate produced according to theSATA method fails to elicit HLA-DR upregulation following 18 hours ofstimulation. FIG. 126E also shows that the BB-11 immunoconjugateproduced according to the SATA method is superior at eliciting HLA-DRupregulation as compared to BB-12 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-11 and BB-12 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 126F shows that the BB-12 immunoconjugate produced according to theSATA method fails to elicit CD14 downregulation following 18 hours ofstimulation as compared to equimolar concentrations of the mixture(CD20+IRM1). FIG. 126F also shows that the BB-11 immunoconjugateproduced according to the SATA method is superior at eliciting CD14downregulation as compared to BB-12 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-11 and BB-12 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 126G also shows that the BB-11 immunoconjugate produced accordingto the SATA method is superior at eliciting CD16 downregulation ascompared to BB-12 and equimolar concentrations of the mixture(CD20+IRM1). It should be noted that BB-11 and BB-12 are constructedwith identical linkers, but have distinct adjuvants.

FIG. 126H shows that the BB-12 immunoconjugate produced according to theSATA method fails to elicit CD40 upregulation following 18 hours ofstimulation as compared to equimolar concentrations of the mixture(CD20+IRM1). FIG. 126H also shows that the BB-11 immunoconjugateproduced according to the SATA method is superior at eliciting CD40upregulation as compared to BB-12 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-11 and BB-12 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 126I shows that the BB-12 immunoconjugate produced according to theSATA method fails to elicit CD86 upregulation following 18 hours ofstimulation as compared to equimolar concentrations of the mixture(CD20+IRM1). FIG. 126I also shows that the BB-11 immunoconjugateproduced according to the SATA method is superior at eliciting CD86upregulation as compared to BB-12 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-11 and BB-12 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 126J shows CD123 expression following 18 hours of stimulation withBB-12. The dashed line indicates the level of CD123 expression onunstimulated cells following 18 hours of incubation.

FIG. 126K shows HLA-DR expression following 18 hours of stimulation withBB-12. The dashed line indicates the level of HLA-DR expression onunstimulated cells following 18 hours of incubation.

FIG. 126L shows CD14 expression following 18 hours of stimulation withBB-12. The dashed line indicates the level of CD14 expression onunstimulated cells following 18 hours of incubation.

FIG. 126M shows CD16 expression following 18 hours of stimulation withBB-12. The dashed line indicates the level of CD16 expression onunstimulated cells following 18 hours of incubation.

FIG. 126N shows CD40 expression following 18 hours of stimulation withBB-12. The dashed line indicates the level of CD40 expression onunstimulated cells following 18 hours of incubation.

FIG. 126O shows CD86 expression following 18 hours of stimulation withBB-12. The dashed line indicates the level of CD86 expression onunstimulated cells following 18 hours of incubation.

FIG. 127A shows a liquid chromatography-mass spectrometry analysis ofthe BB-10 immunoconjugate produced according to the SATA conjugationmethod following overnight deglycosylation with PNGase.

FIG. 127B shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-10.

FIG. 127C shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-10 following overnight deglycosylation with PNGase F.

FIG. 127D shows that the BB-10 immunoconjugate produced according to theSATA method fails to elicit CD123 upregulation following 18 hours ofstimulation. FIG. 127D also shows that the BB-05 immunoconjugateproduced according to the SATA method is superior at eliciting CD123upregulation as compared to BB-10 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-05 and BB-10 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 127E shows that the BB-10 immunoconjugate produced according to theSATA method fails to elicit HLA-DR upregulation following 18 hours ofstimulation. FIG. 127E also shows that the BB-05 immunoconjugateproduced according to the SATA method is superior at eliciting HLA-DRupregulation as compared to BB-10 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-05 and BB-10 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 127F shows that the BB-10 immunoconjugate produced according to theSATA method fails to elicit CD14 downregulation following 18 hours ofstimulation as compared to equimolar concentrations of the mixture(CD20+IRM1). FIG. 127F also shows that the BB-05 immunoconjugateproduced according to the SATA method is superior at eliciting CD14downregulation as compared to BB-10 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-05 and BB-10 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 127G shows that the BB-05 immunoconjugate produced according to theSATA method is superior at eliciting CD16 downregulation as compared toBB-10 and equimolar concentrations of the mixture (CD20+IRM1). It shouldbe noted that BB-05 and BB-10 are constructed with identical linkers,but have distinct adjuvants.

FIG. 127H shows that the BB-10 immunoconjugate produced according to theSATA method fails to elicit CD40 upregulation following 18 hours ofstimulation as compared to equimolar concentrations of the mixture(CD20+IRM1). FIG. 127H also shows that the BB-05 immunoconjugateproduced according to the SATA method is superior at eliciting CD40upregulation as compared to BB-10 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-05 and BB-10 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 127I shows that the BB-10 immunoconjugate produced according to theSATA method fails to elicit CD86 upregulation following 18 hours ofstimulation as compared to equimolar concentrations of the mixture(CD20+IRM1). FIG. 127I also shows that the BB-05 immunoconjugateproduced according to the SATA method is superior at eliciting CD86upregulation as compared to BB-10 and equimolar concentrations of themixture (CD20+IRM1). It should be noted that BB-05 and BB-10 areconstructed with identical linkers, but have distinct adjuvants.

FIG. 127J shows CD123 expression following 18 hours of stimulation withBB-10. The dashed line indicates the level of CD123 expression onunstimulated cells following 18 hours of incubation.

FIG. 127K shows HLA-DR expression following 18 hours of stimulation withBB-10. The dashed line indicates the level of HLA-DR expression onunstimulated cells following 18 hours of incubation.

FIG. 127L shows CD14 expression following 18 hours of stimulation withBB-10. The dashed line indicates the level of CD14 expression onunstimulated cells following 18 hours of incubation.

FIG. 127M shows CD16 expression following 18 hours of stimulation withBB-10. The dashed line indicates the level of CD16 expression onunstimulated cells following 18 hours of incubation.

FIG. 127N shows CD40 expression following 18 hours of stimulation withBB-10. The dashed line indicates the level of CD40 expression onunstimulated cells following 18 hours of incubation.

FIG. 127O shows CD86 expression following 18 hours of stimulation withBB-10. The dashed line indicates the level of CD40 expression onunstimulated cells following 18 hours of incubation.

FIG. 128A shows that the BB-01 immunoconjugate produced according to theBB-01 method elicits superior IL-10 secretion from myeloid cells ascompared to equimolar concentrations of BB-19 produced according tomethods disclosed in U.S. Pat. No. 8,951,528 and unconjugated Rituximabbiosimilar (LGM Pharma) following 18 hours of stimulation.

FIG. 128B shows that the BB-01 immunoconjugate produced according to theBB-01 method elicits superior TNFα secretion from myeloid cells ascompared to equimolar concentrations of BB-19 produced according tomethods disclosed in U.S. Pat. No. 8,951,528 and unconjugated Rituximabbiosimilar (LGM Pharma) following 18 hours of stimulation.

FIG. 128C shows IL-1β secretion from myeloid cells following an 18 hourincubation with equimolar concentrations of unconjugated Rituximabbiosimilar (LGM Pharma) or BB-19 produced according to methods disclosedin U.S. Pat. No. 8,951,528.

FIG. 128D shows TNFα secretion from myeloid cells following an 18 hourincubation with equimolar concentrations of unconjugated Rituximabbiosimilar (LGM Pharma) or BB-19 produced according to methods disclosedin U.S. Pat. No. 8,951,528.

FIG. 128E shows a liquid chromatography-mass spectrometry analysis ofthe BB-19 produced according to methods disclosed in U.S. Pat. No.8,951,528 following overnight deglycosylation with PNGase.

FIG. 128F shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-19 following overnight deglycosylation with PNGase.

FIG. 128G shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-19

FIG. 128H shows that BB-01 produced according to the BB-01 method issuperior at eliciting CD 123 upregulation on myeloid cells as comparedto the BB-19 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 128I shows that BB-01 produced according to the BB-01 method issuperior at eliciting HLA-DR upregulation on myeloid cells as comparedto the BB-19 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 128J shows that BB-01 produced according to the BB-01 method issuperior at eliciting CD14 downregulation on myeloid cells as comparedto the BB-19 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 128K shows that BB-01 produced according to the BB-01 method issuperior at eliciting CD16 downregulation on myeloid cells as comparedto the BB-19 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 128L shows that the BB-19 immunoconjugate produced according tomethods disclosed in U.S. Pat. No. 8,951,528 fails to elicit CD40upregulation following 18 hours of stimulation. FIG. 128L also showsthat the BB-01 immunoconjugate produced according to the BB-01 method issuperior at eliciting CD40 upregulation as compared to BB-19 and theunconjugated Rituximab biosimilar (CD20; LGM Pharma).

FIG. 128M shows that BB-01 produced according to the BB-01 method issuperior at eliciting CD86 upregulation on myeloid cells as compared tothe BB-19 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 128N shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-19 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 128O shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-19 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 128P shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-19 immunoconjugate produced according to themethods disclosed in U.S. Pat. No. 8,951,528 or the unconjugatedRituximab biosimilar (CD20; LGM Pharma).

FIG. 128Q shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-19 immunoconjugate produced according to themethods disclosed in U.S. Pat. No. 8,951,528 or the unconjugatedRituximab biosimilar (CD20; LGM Pharma).

FIG. 128R shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-19 immunoconjugate produced according to themethods disclosed in U.S. Pat. No. 8,951,528 or the unconjugatedRituximab biosimilar (CD20; LGM Pharma).

FIG. 128S shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-19 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 129A shows that the BB-01 immunoconjugate produced according to theBB-01 method elicits superior IL-10 secretion from myeloid cells ascompared to equimolar concentrations of BB-20 produced according to themethods disclosed in U.S. Pat. No. 8,951,528 and unconjugated Rituximabbiosimilar (LGM Pharma) following 18 hours of stimulation.

FIG. 129B shows that the BB-01 immunoconjugate produced according to theBB-01 method elicits superior TNFα secretion from myeloid cells ascompared to equimolar concentrations of BB-20 produced according tomethods disclosed in U.S. Pat. No. 8,951,528 and unconjugated Rituximabbiosimilar (LGM Pharma) following 18 hours of stimulation.

FIG. 129C shows IL-1β secretion from myeloid cells following an 18 hourincubation with equimolar concentrations of unconjugated Rituximabbiosimilar (LGM Pharma) or BB-20 produced according to the methodsdisclosed in U.S. Pat. No. 8,951,528.

FIG. 129D shows TNFα secretion from myeloid cells following an 18 hourincubation with equimolar concentrations of unconjugated Rituximabbiosimilar (LGM Pharma) or BB-20 produced according to the methodsdisclosed in U.S. Pat. No. 8,951,528.

FIG. 129E shows a liquid chromatography-mass spectrometry analysis ofthe BB-20 produced according to methods disclosed in U.S. Pat. No.8,951,528 following overnight deglycosylation with PNGase.

FIG. 129F shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-20 following overnight deglycosylation with PNGase.

FIG. 129G shows a liquid chromatography-mass spectrometry analysis of anunconjugated Rituximab biosimilar (LGM Pharma) that was utilized toproduce BB-20

FIG. 129H shows that BB-01 produced according to the BB-01 method issuperior at eliciting CD123 upregulation on myeloid cells as compared tothe BB-20 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 129I shows that BB-01 produced according to the BB-01 method issuperior at eliciting HLA-DR upregulation on myeloid cells as comparedto the BB-20 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 129J shows that BB-01 produced according to the BB-01 method issuperior at eliciting CD14 downregulation on myeloid cells as comparedto the BB-20 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 129K shows that BB-01 produced according to the BB-01 method issuperior at eliciting CD16 downregulation on myeloid cells as comparedto the BB-20 immunoconjugate produced according to methods disclosed inU.S. Pat. No. 8,951,528 and the unconjugated Rituximab biosimilar (CD20;LGM Pharma) following 18 hours of stimulation.

FIG. 129L shows that the BB-20 immunoconjugate produced according tomethods disclosed in U.S. Pat. No. 8,951,528 fails to elicit CD40upregulation following 18 hours of stimulation. FIG. 129L also showsthat the BB-01 immunoconjugate produced according to the BB-01 method issuperior at eliciting CD40 upregulation as compared to BB-20 and theunconjugated Rituximab biosimilar (CD20; LGM Pharma).

FIG. 129M shows that the BB-20 immunoconjugate produced according tomethods disclosed in U.S. Pat. No. 8,951,528 fails to elicit CD86upregulation following 18 hours of stimulation. FIG. 129M also showsthat the BB-01 immunoconjugate produced according to the BB-01 method issuperior at eliciting CD86 upregulation as compared to BB-20 and theunconjugated Rituximab biosimilar (CD20; LGM Pharma).

FIG. 129N shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-20 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 129O shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-20 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 129P shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-20 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 129Q shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-20 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 129R shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-20 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 129S shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-20 immunoconjugate produced according to methodsdisclosed in U.S. Pat. No. 8,951,528 or the unconjugated Rituximabbiosimilar (CD20; LGM Pharma).

FIG. 130A shows CD14 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of bovine serum albumin (BSA)or BSA immunoconjugate (BSA-Compound 1) produced according the BB-37 TFPmethod.

FIG. 130B shows CD16 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of bovine serum albumin (BSA)or BSA immunoconjugate (BSA-Compound 1) produced according the BB-37 TFPmethod.

FIG. 130C shows CD123 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of bovine serum albumin (BSA)or BSA immunoconjugate (BSA-Compound 1) produced according the BB-37 TFPmethod.

FIG. 130D shows CD40 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of bovine serum albumin (BSA)or BSA immunoconjugate (BSA-Compound 1) produced according the BB-37 TFPmethod.

FIG. 130E shows CD86 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of bovine serum albumin (BSA)or BSA immunoconjugate (BSA-Compound 1) produced according the BB-37 TFPmethod.

FIG. 130F shows HLR-DR expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of bovine serum albumin (BSA)or BSA immunoconjugate (BSA-Compound 1) produced according the BB-37 TFPmethod.

FIG. 130G shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of bovine serum albumin (BSA)or BSA immunoconjugate (BSA-Compound 1) produced according the BB-37 TFPmethod.

FIG. 130H shows LC-MS of naked BSA-M.

FIG. 131A shows CD14 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of keyhole limpet hemocyanin(KLH) or KLH immunoconjugate (KLH-Compound 1) produced according theBB-17 TFP method.

FIG. 131B shows CD16 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of keyhole limpet hemocyanin(KLH) or KLH immunoconjugate (KLH-Compound 1) produced according theBB-17 TFP method.

FIG. 131C shows CD123 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of keyhole limpet hemocyanin(KLH) or KLH immunoconjugate (KLH-Compound 1) produced according theBB-17 TFP method.

FIG. 131D shows CD40 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations of keyhole limpet hemocyanin(KLH) or KLH immunoconjugate (KLH-Compound 1) produced according theBB-17 TFP method.

FIG. 131E shows CD86 expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations keyhole limpet hemocyanin(KLH) or KLH immunoconjugate (KLH-Compound 1) produced according theBB-17 TFP method.

FIG. 131F shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with equimolar concentrations keyhole limpet hemocyanin(KLH) or KLH immunoconjugate (KLH-Compound 1) produced according theBB-17 TFP method.

FIG. 132A shows that Enbrel (Amgen) and Enbrel immunoconjugate (BB-01Enbrel), produced using the BB-01 conjugation method, show comparablereactivity with anti-human IgG detection antibody when captured onanti-human IgG coated ELISA plates.

FIG. 132B shows that Enbrel immunoconjugate (BB-01 Enbrel), producedusing the BB-01 conjugation method, but not Enbrel (Amgen), shows strongreactivity with the anti-Compound 1 antibody following capture on ananti-human IgG coated ELISA plate.

FIG. 132C shows that Cetuximab (Imclone/Lilly) and Cetuximabimmunoconjugate (BB-01 Cetuximab), produced using the BB-01 conjugationmethod, show comparable reactivity with anti-human IgG detectionantibody when captured on anti-human IgG coated ELISA plates.

FIG. 132D shows that Cetuximab immunoconjugate (BB-01 Cetuximab),produced using the BB-01 conjugation method, but not Cetuximab(Imclone/Lilly), shows strong reactivity with the anti-Compound 1antibody following capture on an anti-human IgG coated ELISA plate.

FIG. 132E shows that Ipilimumab (BMS) and Ipilimumab immunoconjugate(BB-01 Ipilimumab), produced using the BB-01 conjugation method, showcomparable reactivity with anti-human IgG detection antibody whencaptured on anti-human IgG coated ELISA plates.

FIG. 132F shows that Ipilimumab immunoconjugate (BB-01 Ipilimumab),produced using the BB-01 conjugation method, but not Ipilimumab (BMS),shows strong reactivity with the anti-Compound 1 antibody followingcapture on an anti-human IgG coated ELISA plate.

FIG. 132G shows that Obinutuzumab (Roche) and Obinutuzumabimmunoconjugate (BB-01 Obinutuzumab), produced using the BB-01conjugation method, show comparable reactivity with anti-human IgGdetection antibody when captured on anti-human IgG coated ELISA plates.

FIG. 132H shows that Obinutuzumab immunoconjugate (BB-01 Obinutuzumab),produced using the BB-01 conjugation method, but not Obinutuzumab(Roche), shows strong reactivity with the anti-Compound 1 antibodyfollowing capture on an anti-human IgG coated ELISA plate.

FIG. 132I shows that Rituximab (Roche) and Rituximab immunoconjugate(BB-01 Rituximab), produced using the BB-01 conjugation method, showcomparable reactivity with anti-human IgG detection antibody whencaptured on anti-human IgG coated ELISA plates.

FIG. 132J shows that Rituximab immunoconjugate (BB-01 Rituximab),produced using the BB-01 conjugation method, but not Rituximab (Roche),shows strong reactivity with the anti-Compound 1 antibody followingcapture on an anti-human IgG coated ELISA plate.

FIG. 132K shows that Anti-Dectin 2 Antibody (Biorad MCA2415) andAnti-Dectin 2 immunoconjugate (BB-01 Anti-Dectin 2), produced using theBB-01 conjugation method, show comparable reactivity with IgG detectionantibody when coated onto an ELISA plate.

FIG. 132L shows that Anti-Dectin 2 immunoconjugate (BB-01 Anti-Dectin2), produced using the BB-01 conjugation method, but not Anti-Dectin 2Antibody (Biorad MCA2415), shows strong reactivity with theanti-Compound 1 antibody by ELISA assay.

FIG. 133A shows that Rituximab immunoconjugates (Rituximab BB-01) retainbinding activity for CD16a. Binding was assayed by ELISA as described inExample 29. Compounds show are Rituximab, aglycosyl Rituximab,(Invivogen hcd20-mab12), or Rituximab immunoconjugates (RituximabBB-01). DAR levels on Rituximab conjugates were as follows: BB-01, 1.1;BB-14, 2.0; BB-36 low DAR, 1.4; BB36 high DAR, 2.8; BB37 low DAR, 1.7;BB-37 high DAR, 2.6. The Y axis shows the fraction of maximal OD signalat highest concentration for each sample. The aglycosyl mutant ofRituximab shows diminished binding, consistent with the role ofglycosylation in effector function.

FIG. 133B shows that Rituximab (Roche) and Rituximab immunoconjugates(BB-01 Rituximab), produced using the BB-01 conjugation method, showcomparable binding to CD64 immobilized on ELISA plates. Rituximab hadbeen deglycosylated used PNGase F shows impaired binding to CD64.

FIG. 133C shows that Rituximab and a Rituximab immunoconjugate(Rituximab BB-37) bind to protein A. Duplicate samples were subjected topull down using protein A sepharose. No unbound Rituximab or RituximabBB-37 was detected in the pull down supernatants. There is considerableoverlap of protein A and FcRN binding sites on IgG. Therefore,preservation of protein A binding in Rituximab BB-37 suggestspreservation of FcRN binding.

FIG. 134A shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-48 immunoconjugate produced according to theBB-48 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 134B shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-48 immunoconjugate produced according to theBB-48 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 134C shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-48 immunoconjugate produced according to theBB-48 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 134D shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-48 immunoconjugate produced according to theBB-48 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 134E shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-48 immunoconjugate produced according to theBB-48 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 134F shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-48 immunoconjugate produced according to theBB-48 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 134G shows LC-MS for BB-48 immunoconjugate produced according tothe BB-48 method.

FIG. 135A shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-49 immunoconjugate produced according to theBB-49 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 135B shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-49 immunoconjugate produced according to theBB-49 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 135C shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-49 immunoconjugate produced according to theBB-49 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 135D shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-49 immunoconjugate produced according to theBB-49 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 135E shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-49 immunoconjugate produced according to theBB-49 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 135F shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-49 immunoconjugate produced according to theBB-49 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 135G shows LC-MS for BB-49 immunoconjugate produced according tothe BB-49 method.

FIG. 136A shows CD123 expression on myeloid cells following 18 hours ofstimulation with the BB-50 immunoconjugate produced according to theBB-50 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 136B shows HLA-DR expression on myeloid cells following 18 hours ofstimulation with the BB-50 immunoconjugate produced according to theBB-50 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 136C shows CD14 expression on myeloid cells following 18 hours ofstimulation with the BB-50 immunoconjugate produced according to theBB-50 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 136D shows CD16 expression on myeloid cells following 18 hours ofstimulation with the BB-50 immunoconjugate produced according to theBB-50 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 136E shows CD40 expression on myeloid cells following 18 hours ofstimulation with the BB-50 immunoconjugate produced according to theBB-50 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 136F shows CD86 expression on myeloid cells following 18 hours ofstimulation with the BB-50 immunoconjugate produced according to theBB-50 method or the unconjugated Rituximab biosimilar (CD20; Alphamab).

FIG. 136G shows LC-MS for BB-50 immunoconjugate produced according tothe BB-50 method.

FIG. 137A shows that the BB-01 immunoconjugate produced according to theBB-01 SATA method (Rituximab Boltbody) is superior at eliciting CD14downregulation on myeloid cells as compared to the rituximabimmunoconjugate conjugated through the interchain disulfides residuesfollowing TCEP reduction via SMCC-Cmpd1 (Rituximab-Cys-Cmpd1). Data wereobtained following 18-hour incubation with either Rituximab Boltbody orRituximab-Cys-Cmpd1.

FIG. 137B shows that the BB-01 immunoconjugate produced according to theBB-01 SATA method (Rituximab Boltbody) is superior at eliciting CD16downregulation on myeloid cells as compared to the rituximabimmunoconjugate conjugated through the interchain disulfides residuesfollowing TCEP reduction via SMCC-Cmpd1 (Rituximab-Cys-Cmpd1). Data wereobtained following 18-hour incubation with either Rituximab Boltbody orRituximab-Cys-Cmpd1.

FIG. 137C shows that the BB-01 immunoconjugate produced according to theBB-01 SATA method (Rituximab Boltbody) is superior at eliciting CD40upregulation on myeloid cells as compared to the rituximabimmunoconjugate conjugated through the interchain disulfides residuesfollowing TCEP reduction via SMCC-Cmpd1 (Rituximab-Cys-Cmpd1). Data wereobtained following 18-hour incubation with either Rituximab Boltbody orRituximab-Cys-Cmpd1.

FIG. 137D shows that the BB-01 immunoconjugate produced according to theBB-01 SATA method (Rituximab Boltbody) is superior at eliciting CD86upregulation on myeloid cells as compared to the rituximabimmunoconjugate conjugated through the interchain disulfides residuesfollowing TCEP reduction via SMCC-Cmpd1 (Rituximab-Cys-Cmpd1). Data wereobtained following 18-hour incubation with either Rituximab Boltbody orRituximab-Cys-Cmpd1.

FIG. 137E shows that the BB-01 immunoconjugate produced according to theBB-01 SATA method (Rituximab Boltbody) is superior at eliciting CD123upregulation on myeloid cells as compared to the rituximabimmunoconjugate conjugated through the interchain disulfides residuesfollowing TCEP reduction via SMCC-Cmpd1 (Rituximab-Cys-Cmpd1). Data wereobtained following 18-hour incubation with either Rituximab Boltbody orRituximab-Cys-Cmpd1.

FIG. 137F shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab (Roche) following reduction with TCEP that wasutilized to produce the rituximab-cys-cmpd1 immunoconjugate.

FIG. 137G shows a liquid chromatography-mass spectrometry analysis ofthe light chain of unconjugated rituximab (Roche) following reductionwith TCEP that was utilized to produce the rituximab-cys-cmpd1immunoconjugate.

FIG. 137H shows a liquid chromatography-mass spectrometry analysis ofthe heavy chain of the rituximab-cys-cmpd1 immunoconjugate.

FIG. 137I shows a liquid chromatography-mass spectrometry analysis ofthe heavy chain of the rituximab-cys-cmpd1 immunoconjugate.

FIG. 138A shows the DNA sequence for the vector encoding the heavy chainof wildtype rituximab.

FIG. 138B shows the DNA sequence for the vector encoding the kappa lightchain of rituximab with the V205C mutation (denoted using kabatnumbering).

FIG. 138C shows the vector map for the pFUSE-CHIg-hG1 cloning plasmid(Invivogen, pfuse-hchg1) encoding for the wildtype rituximab IgG1 heavychain.

FIG. 138D shows the vector map for the pFUSE2-CLIg-hK cloning plasmid(Invivogen, pfuse2-hclk) engineered to encode the V205C mutation in theconstant region of the rituximab Ig kappa light chain.

FIG. 138E shows the structure of the rituximab-V205C immunoconjugateproduced by direct linkage of compound 1-SMCC to the engineered cysteineresidues as described in FIG. 138D.

FIG. 138F shows a liquid chromatography-mass spectrometry analysis ofunconjugated rituximab containing the V205C mutation that was utilizedto produce the rituximab-V205C immunoconjugate.

FIG. 138G shows a liquid chromatography-mass spectrometry analysis ofthe rituximab-V205C immunoconjugate produced by direct linkage ofcompound 1-SMCC to the engineered cysteine residues as described in FIG.138D.

FIG. 139 shows synthetic Scheme 3 of Example 2.

FIG. 14O shows synthetic Scheme 14 of Example 8.

FIG. 14I shows synthetic Scheme 15 of Example 9.

FIG. 142 shows synthetic Scheme 16 of Example 10.

FIG. 143 shows synthetic Scheme 17 of Example 11.

FIG. 144 shows synthetic Scheme 20 of Example 13.

FIG. 145 shows synthetic Scheme 21 of Example 14.

FIG. 146 shows synthetic Scheme 22 of Example 15.

FIG. 147 shows synthetic Scheme 23 of Example 16.

FIG. 148 shows synthetic Scheme 24 of Example 17.

FIG. 149 shows synthetic Scheme 25 of Example 18.

FIG. 15O shows synthetic Scheme 27 of Example 20.

FIG. 15I shows synthetic Scheme 28 of Example 21.

DETAILED DESCRIPTION OF THE INVENTION General

The invention provides antibody-adjuvant immunoconjugates having anumber of advantages including: antibodies that promoteantibody-dependent cellular cytotoxicity, antibody-dependent cellularphagocytosis and antibodies that block the actions of cancer producedproteins that act as immune checkpoint molecules, adjuvants that promotedendritic cell activation and T cell proliferation, and covalentlinkages between antibody and adjuvant that promote anti-tumor efficacy.For example, in some cases human monocytes undergo DC differentiationfollowing overnight stimulation with immunoconjugates of the invention,whereas DC differentiation protocols with known stimulants (e.g., GM-CSFand IL-4) require much longer periods. Immunoconjugate-activated cellsexpress higher amounts (e.g., in some cases several fold higher amounts)of co-stimulatory molecules and inflammatory cytokines than isachievable with known stimulants.

As demonstrated herein, immunoconjugates are quantitatively andqualitatively more effective at eliciting immune activation thannon-covalently attached antibody-adjuvant mixtures. Further, asdemonstrated herein, antibody-adjuvant immunoconjugates linked accordingto the present invention are much more effective than other knownimmunoconjugates. For example, immunoconjugates are disclosed in U.S.Pat. No. 8,951,528. However, these immunoconjugates fail to effectivelyactivate myeloid cells (see for example, FIGS. 128A-129S). Anotherpublication, US Patent Application Publication 2017/0158772 disclosesimmunoconjugates, as well. The immunoconjugates disclosed therein alsodo not effectively activate myeloid cells as seen in FIGS. 67A-68P.International Patent Application Publication WO 2015/103987 A1 shows inclaim 1 an immunoconjugate attachment site to an adjuvant (resiquimod)in a location that, through experimentation, inactivates the adjuvantand results in negligible myeloid activation. The publication alsoindicates that conjugation of the linker-adjuvant to the antibody occursthrough cysteine hinge residues (thioether linkages) (WO 2015/103987,paragraphs 0273-0273) following reduction of the antibody with an excessof DTT. Through experimentation, this mode of conjugation prevents theimmunoconjugate from effectively activating myeloid cells (see FIGS.67A-68P and 137A-1371). In contrast, the immunoconjugates of theinvention provide superior biological activity as seen, for example, inFIGS. 67G-K, 128A-12M, 129E-129, and 137A-137I.

Finally, systemic administration of the adjuvant-antibody conjugatesallows for the simultaneous targeting of the primary tumor andassociated metastases without the need for intra-tumoral injections andsurgical resection.

As demonstrated by FIGS. 1-138G, numerous immunoconjugates were createdand assayed in accordance with the invention and other sources.

Definitions

As used herein, the term “immunoconjugate” refers to an antibodyconstruct, or antibody, that is covalently bonded to a non-naturallyoccurring chemical moiety as described herein. The terms“immunoconjugate,” “antibody-adjuvant immunoconjugate,” “AAC,” and“Boltbody” are used interchangeably herein.

As used herein, the phrase “antibody construct” refers to polypeptidecomprising an antigen binding domain and an Fc domain. An antibodyconstruct can comprise an antibody.

As used herein, the phrase “antigen binding domain” refers to a protein,or a portion of a protein, that specifically binds a specified antigen(e.g., a paratope). For example, that portion of an antigen-bindingprotein that contains the amino acid residues that interact with anantigen and confer on the antigen-binding protein its specificity andaffinity for the antigen.

As used herein, the phrase “Fc domain” refers to the fragmentcrystallizable region, or the tail region of an antibody. The Fc domaininteracts with Fc receptors on cells' surfaces.

As used herein, the phrase “targeting binding domain” refers to aprotein, or a portion of a protein, that specifically binds a secondantigen that is distinct from the antigen bound by the antigen bindingdomain of the immunoconjugates. The targeting binding domain can beconjugated to the antibody construct at a C-terminal end of the Fcdomain.

As used herein, the term “antibody” refers to a polypeptide comprisingan antigen binding region (including the complementarity determiningregion (CDRs)) from an immunoglobulin gene or fragments thereof thatspecifically binds and recognizes an antigen. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon, and mu constant region genes, as well as numerousimmunoglobulin variable region genes.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively.

IgG antibodies are large molecules of about 150 kDa composed of fourpeptide chains. IgG antibodies contain two identical class γ heavychains of about 50 kDa and two identical light chains of about 25 kDa,thus a tetrameric quaternary structure. The two heavy chains are linkedto each other and to a light chain each by disulfide bonds. Theresulting tetramer has two identical halves, which together form theY-like shape. Each end of the fork contains an identical antigen bindingsite. There are four IgG subclasses (IgG1, 2, 3, and 4) in humans, namedin order of their abundance in serum (IgG1 being the most abundant).Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

Dimeric IgA antibodies are around 320 kDa. IgA has two subclasses (IgA1and IgA2) and can be produced as a monomeric as well as a dimeric form.The IgA dimeric form (secretory or sIgA) is the most abundant.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into a Fab′ monomer. The Fab′ monomer is essentially Fab with partof the hinge region (see, Fundamental Immunology (Paul ed., 7e ed.2012). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature, 348: 552-554(1990)).

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. “Antibody fragment,” and allgrammatical variants thereof, as used herein are defined as a portion ofan intact antibody comprising the antigen binding site or variableregion of the intact antibody, wherein the portion is free of theconstant heavy chain domains (i.e. CH2, CH3, and CH4, depending onantibody isotype) of the Fc region of the intact antibody. Examples ofantibody fragments include Fab, Fab′, Fab′-SH, F(ab′)₂, and Fvfragments; diabodies; any antibody fragment that is a polypeptide havinga primary structure consisting of one uninterrupted sequence ofcontiguous amino acid residues (referred to herein as a “single-chainantibody fragment” or “single chain polypeptide”), including withoutlimitation (1) single-chain Fv (scFv) molecules; (2) single chainpolypeptides containing only one light chain variable domain, or afragment thereof that contains the three CDRs of the light chainvariable domain, without an associated heavy chain moiety; (3) singlechain polypeptides containing only one heavy chain variable region, or afragment thereof containing the three CDRs of the heavy chain variableregion, without an associated light chain moiety; (4) nanobodiescomprising single Ig domains from non-human species or other specificsingle-domain binding modules; and (5) multispecific or multivalentstructures formed from antibody fragments. In an antibody fragmentcomprising one or more heavy chains, the heavy chain(s) can contain anyconstant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fcregion of an intact antibody, and/or can contain any hinge regionsequence found in an intact antibody, and/or can contain a leucinezipper sequence fused to or situated in the hinge region sequence or theconstant domain sequence of the heavy chain(s).

As used herein, the term “biosimilar” in reference to a biologicalproduct, means that the biological product is highly similar to thereference product notwithstanding minor differences in clinicallyinactive components, and there are no clinically meaningful differencesbetween the biological product and the reference product in terms of thesafety, purity, and potency of the product.

As used herein, the term “epitope” means any antigenic determinant on anantigen to which the antigen-binding site, also referred to as theparatope, of an antibody binds. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

As used herein, the term “adjuvant” refers to a substance capable ofeliciting an immune response in a subject exposed to the adjuvant.

As used herein, the term “adjuvant moiety” refers to an adjuvant that iscovalently bonded to an antibody as described herein. The adjuvantmoiety can elicit the immune response while bonded to the antibody, orafter cleavage (e.g., enzymatic cleavage) from the antibody followingadministration of an immunoconjugate to the subject.

As used herein, the terms “Pattern recognition receptor” and “PRR” referto any member of a class of conserved mammalian proteins which recognizepathogen-associated molecular patterns (PAMPs) or damage-associatedmolecular patterns (DAMPs), and act as key signaling elements in innateimmunity. Pattern recognition receptors are divided into membrane-boundPRRs, cytoplasmic PRRs, and secreted PRRs. Examples of membrane-boundPRRs include Toll-like receptors (TLRs) and C-type lectin receptors(CLRs). Examples of cytoplasmic PRRs include NOD-like receptors (NLRs)and Rig-I-like receptors (RLRs).

As used herein, the terms “Toll-like receptor” and “TLR” refer to anymember of a family of highly-conserved mammalian proteins whichrecognize pathogen-associated molecular patterns and act as keysignaling elements in innate immunity. TLR polypeptides share acharacteristic structure that includes an extracellular domain that hasleucine-rich repeats, a transmembrane domain, and an intracellulardomain that is involved in TLR signaling.

The terms “Toll-like receptor 1” and “TLR1” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR1 sequence, e.g.,GenBank accession number AAY85643 for human TLR1 polypeptide, or GenBankaccession number AAG37302 for murine TLR1 polypeptide.

The terms “Toll-like receptor 2” and “TLR2” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR2 sequence, e.g.,GenBank accession number AAY85648 for human TLR2 polypeptide, or GenBankaccession number AAD49335 for murine TLR2 polypeptide.

The terms “Toll-like receptor 3” and “TLR3” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR3 sequence, e.g.,GenBank accession number AAC34134 for human TLR3 polypeptide, or GenBankaccession number AAK26117 for murine TLR3 polypeptide.

The terms “Toll-like receptor 4” and “TLR4” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR4 sequence, e.g.,GenBank accession number AAY82270 for human TLR4 polypeptide, or GenBankaccession number AAD29272 for murine TLR4 polypeptide.

The terms “Toll-like receptor 5” and “TLR5” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR5 sequence, e.g.,GenBank accession number ACM69034 for human TLR5 polypeptide, or GenBankaccession number AAF65625 for murine TLR5 polypeptide.

The terms “Toll-like receptor 6” and “TLR6” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR6 sequence, e.g.,GenBank accession number ABY67133 for human TLR6 polypeptide, or GenBankaccession number AAG38563 for murine TLR6 polypeptide.

The terms “Toll-like receptor 7” and “TLR7” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR7 sequence, e.g.,GenBank accession number AAZ99026 for human TLR7 polypeptide, or GenBankaccession number AAK62676 for murine TLR7 polypeptide.

The terms “Toll-like receptor 8” and “TLR8” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR8 sequence, e.g.,GenBank accession number AAZ95441 for human TLR8 polypeptide, or GenBankaccession number AAK62677 for murine TLR8 polypeptide.

The terms “Toll-like receptor 7/8” and “TLR7/8” refer to nucleic acidsor polypeptides that are both TLR7 agonists and TLR8 agonists.

The terms “Toll-like receptor 9” and “TLR9” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR9 sequence, e.g.,GenBank accession number AAF78037 for human TLR9 polypeptide, or GenBankaccession number AAK28488 for murine TLR9 polypeptide.

The terms “Toll-like receptor 10” and “TLR10” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR10 sequence, e.g.,GenBank accession number AAK26744 for human TLR10 polypeptide.

The terms “Toll-like receptor 11” and “TLR11” refer to nucleic acids orpolypeptides sharing at least 70%; 80%, 90%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a publicly-available TLR11 sequence, e.g.,GenBank accession number AAS83531 for murine TLR11 polypeptide.

A “TLR agonist” is a substance that binds, directly or indirectly, to aTLR (e.g., TLR7 and/or TLR8) to induce TLR signaling. Any detectabledifference in TLR signaling can indicate that an agonist stimulates oractivates a TLR. Signaling differences can be manifested, for example,as changes in the expression of target genes, in the phosphorylation ofsignal transduction components, in the intracellular localization ofdownstream elements such as NK-κB, in the association of certaincomponents (such as IRAK) with other proteins or intracellularstructures, or in the biochemical activity of components such as kinases(such as MAPK).

As used herein, the term “amino acid” refers to any monomeric unit thatcan be incorporated into a peptide, polypeptide, or protein. Amino acidsinclude naturally-occurring a-amino acids and their stereoisomers, aswell as unnatural (non-naturally occurring) amino acids and theirstereoisomers. “Stereoisomers” of a given amino acid refer to isomershaving the same molecular formula and intramolecular bonds but differentthree-dimensional arrangements of bonds and atoms (e.g., an L-amino acidand the corresponding D-amino acid).

Naturally-occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, y-carboxyglutamate, and O-phosphoserine.Naturally-occurring a-amino acids include, without limitation, alanine(Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu),phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile),arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met),asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser),threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), andcombinations thereof. Stereoisomers of a naturally-occurring a-aminoacids include, without limitation, D-alanine (D-Ala), D-cysteine(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu),D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile),D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine(D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln),D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan(D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

Unnatural (non-naturally occurring) amino acids include, withoutlimitation, amino acid analogs, amino acid mimetics, synthetic aminoacids, N-substituted glycines, and N-methyl amino acids in either the L-or D-configuration that function in a manner similar to thenaturally-occurring amino acids. For example, “amino acid analogs” canbe unnatural amino acids that have the same basic chemical structure asnaturally-occurring amino acids (i.e., a carbon that is bonded to ahydrogen, a carboxyl group, an amino group) but have modified side-chaingroups or modified peptide backbones, e.g., homoserine, norleucine,methionine sulfoxide, methionine methyl sulfonium. “Amino acid mimetics”refer to chemical compounds that have a structure that is different fromthe general chemical structure of an amino acid, but that functions in amanner similar to a naturally-occurring amino acid. Amino acids may bereferred to herein by either the commonly known three letter symbols orby the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission.

As used herein, the term “immune checkpoint inhibitors” refers to anymodulator that inhibits the activity of the immune checkpoint molecule.Immune checkpoint inhibitors can include, but are not limited to, immunecheckpoint molecule binding proteins, small molecule inhibitors,antibodies, antibody-derivatives (including Fc fusions, Fab fragmentsand scFvs), antibody-drug conjugates, antisense oligonucleotides, siRNA,aptamers, peptides and peptide mimetics.

As used herein, the term “linking moiety” refers to a functional groupthat covalently bonds two or more moieties in a compound or material.For example, the linking moiety can serve to covalently bond an adjuvantmoiety to an antibody in an immunoconjugate.

Useful bonds for connecting linking moieties to proteins and othermaterials include, but are not limited to, amides, amines, esters,carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, andthioureas. A “divalent” linking moiety contains two points of attachmentfor linking two functional groups; polyvalent linking moieties can haveadditional points of attachment for linking further functional groups.For example, divalent linking moieties include divalent polymer moietiessuch as divalent poly(ethylene glycol), divalent poly(propylene glycol),and divalent poly(vinyl alcohol).

As used herein, when the term “optionally present” is used to refer to achemical structure (e.g., “R” or “Q”), if that chemical structure is notpresent, the bond originally made to the chemical structure is madedirectly to the adjacent atom.

As used herein, the term “linker” refers to a functional group thatcovalently bonds two or more moieties in a compound or material. Forexample, the linker can serve to covalently bond an adjuvant moiety toan antibody construct in an immunoconjugate.

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. Alkyl can include any number of carbons, such as C₁₋₂, C₁₋₃,C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄,C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. For example, C₁₋₆ alkyl includes, butis not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can alsorefer to alkyl groups having up to 30 carbons atoms, such as, but notlimited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can besubstituted or unsubstituted. “Substituted alkyl” groups can besubstituted with one or more groups selected from halo, hydroxy, amino,oxo (═O), alkylamino, amido, acyl, nitro, cyano, and alkoxy. The term“alkylene” refers to a divalent alkyl radical.

As used herein, the term “heteroalkyl” refers to an alkyl group asdescribed herein, wherein one or more carbon atoms are optionally andindependently replaced with heteroatom selected from N, O, and S. Theterm “heteroalkylene” refers to a divalent heteroalkyl radical.

As used herein, the term “carbocycle” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic, or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated. Carbocycles can include any number of carbons, such as C₃₋₆,C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, C₆₋₈, C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂.Saturated monocyclic carbocyclic rings include, for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl.Saturated bicyclic and polycyclic carbocyclic rings include, forexample, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene andadamantane. Carbocyclic groups can also be partially unsaturated, havingone or more double or triple bonds in the ring. Representativecarbocyclic groups that are partially unsaturated include, but are notlimited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3-and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene,cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, andnorbornadiene.

Unsaturated carbocyclic groups also include aryl groups. The term “aryl”refers to an aromatic ring system having any suitable number of ringatoms and any suitable number of rings. Aryl groups can include anysuitable number of ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ringmembers. Aryl groups can be monocyclic, fused to form bicyclic ortricyclic groups, or linked by a bond to form a biaryl group.Representative aryl groups include phenyl, naphthyl and biphenyl. Otheraryl groups include benzyl, having a methylene linking group. Some arylgroups have from 6 to 12 ring members, such as phenyl, naphthyl orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl.

A “divalent” carbocycle refers to a carbocyclic group having two pointsof attachment for covalently linking two moieties in a molecule ormaterial. Carbocycles can be substituted or unsubstituted. “Substitutedcarbocycle” groups can be substituted with one or more groups selectedfrom halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, andalkoxy.

As used herein, the term “heterocycle” refers to heterocycloalkyl groupsand heteroaryl groups. “Heteroaryl,” by itself or as part of anothersubstituent, refers to a monocyclic or fused bicyclic or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5of the ring atoms are a heteroatom such as N, O or S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can be oxidized to form moieties such as, but notlimited to, —S(O)— and —S(O)₂—. Heteroaryl groups can include any numberof ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitablenumber of heteroatoms can be included in the heteroaryl groups, such as1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2to 5, 3 to 4, or 3 to 5. The heteroaryl group can include groups such aspyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine,pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers),thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Theheteroaryl groups can also be fused to aromatic ring systems, such as aphenyl ring, to form members including, but not limited to,benzopyrroles such as indole and isoindole, benzopyridines such asquinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine(quinazoline), benzopyridazines such as phthalazine and cinnoline,benzothiophene, and benzofuran. Other heteroaryl groups includeheteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groupscan be substituted or unsubstituted. “Substituted heteroaryl” groups canbe substituted with one or more groups selected from halo, hydroxy,amino, oxo (═O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

Heteroaryl groups can be linked via any position on the ring. Forexample, pyrrole includes 1-, 2- and 3-pyrrole, pyridine includes 2-, 3-and 4-pyridine, imidazole includes 1-, 2-, 4- and 5-imidazole, pyrazoleincludes 1-, 3-, 4- and 5-pyrazole, triazole includes 1-, 4- and5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes2-, 4-, 5- and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine,1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-,5- and 6-triazine, 1,3,5-triazine includes 2-triazine, thiopheneincludes 2- and 3-thiophene, furan includes 2- and 3-furan, thiazoleincludes 2-, 4- and 5-thiazole, isothiazole includes 3-, 4- and5-isothiazole, oxazole includes 2-, 4- and 5-oxazole, isoxazole includes3-, 4- and 5-isoxazole, indole includes 1-, 2- and 3-indole, isoindoleincludes 1- and 2-isoindole, quinoline includes 2-, 3- and 4-quinoline,isoquinoline includes 1-, 3- and 4-isoquinoline, quinazoline includes 2-and 4-quinoazoline, cinnoline includes 3- and 4-cinnoline,benzothiophene includes 2- and 3-benzothiophene, and benzofuran includes2- and 3-benzofuran.

“Heterocyclyl,” by itself or as part of another substituent, refers to asaturated ring system having from 3 to 12 ring members and from 1 to 4heteroatoms of N, O and S. Additional heteroatoms can also be useful,including, but not limited to, B, Al, Si and P. The heteroatoms can beoxidized to form moieties such as, but not limited to, —S(O)— and—S(O)₂—. Heterocyclyl groups can include any number of ring atoms, suchas, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatomscan be included in the heterocyclyl groups, such as 1, 2, 3, or 4, or 1to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. The heterocyclyl groupcan include groups such as aziridine, azetidine, pyrrolidine,piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine,piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane,tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane,thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran),oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane,dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Theheterocyclyl groups can also be fused to aromatic or non-aromatic ringsystems to form members including, but not limited to, indoline.Heterocyclyl groups can be unsubstituted or substituted. “Substitutedheterocyclyl” groups can be substituted with one or more groups selectedfrom halo, hydroxy, amino, oxo (═O), alkylamino, amido, acyl, nitro,cyano, and alkoxy.

Heterocyclyl groups can be linked via any position on the ring. Forexample, aziridine can be 1- or 2-aziridine, azetidine can be 1- or2-azetidine, pyrrolidine can be 1-, 2- or 3-pyrrolidine, piperidine canbe 1-, 2-, 3- or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or4-pyrazolidine, imidazolidine can be 1-, 2-, 3- or 4-imidazolidine,piperazine can be 1-, 2-, 3- or 4-piperazine, tetrahydrofuran can be 1-or 2-tetrahydrofuran, oxazolidine can be 2-, 3-, 4- or 5-oxazolidine,isoxazolidine can be 2-, 3-, 4- or 5-isoxazolidine, thiazolidine can be2-, 3-, 4- or 5-thiazolidine, isothiazolidine can be 2-, 3-, 4- or5-isothiazolidine, and morpholine can be 2-, 3- or 4-morpholine.

As used herein, the terms “halo” and “halogen,” by themselves or as partof another substituent, refer to a fluorine, chlorine, bromine, oriodine atom.

As used herein, the term “carbonyl,” by itself or as part of anothersubstituent, refers to —C(O)—, i.e., a carbon atom double-bonded tooxygen and bound to two other groups in the moiety having the carbonyl.

As used herein, the term “amino” refers to a moiety —NR₃, wherein each Rgroup is H or alkyl. An amino moiety can be ionized to form thecorresponding ammonium cation.

As used herein, the term “hydroxy” refers to the moiety —OH.

As used herein, the term “cyano” refers to a carbon atom triple-bondedto a nitrogen atom (i.e., the moiety —C≡N).

As used herein, the term “carboxy” refers to the moiety —C(O)OH. Acarboxy moiety can be ionized to form the corresponding carboxylateanion.

As used herein, the term “amido” refers to a moiety —NRC(O)R or—C(O)NR₂, wherein each R group is H or alkyl.

As used herein, the term “nitro” refers to the moiety —NO₂.

As used herein, the term “oxo” refers to an oxygen atom that isdouble-bonded to a compound (i.e., O═).

As used herein, the terms “treat,” “treatment,” and “treating” refer toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., cognitive impairment), includingany objective or subjective parameter such as abatement; remission;diminishing of symptoms or making the symptom, injury, pathology orcondition more tolerable to the patient; reduction in the rate ofsymptom progression; decreasing the frequency or duration of the symptomor condition; or, in some situations, preventing the onset of thesymptom. The treatment or amelioration of symptoms can be based on anyobjective or subjective parameter; including, e.g., the result of aphysical examination.

As used herein, the term “cancer” refers to conditions including solidcancers, lymphomas, and leukemias. Examples of different types of cancerinclude, but are not limited to, lung cancer (e.g., non-small cell lungcancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer,liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renal cellcarcinoma), bladder cancer, breast cancer, thyroid cancer, pleuralcancer, pancreatic cancer, uterine cancer, cervical cancer, testicularcancer, anal cancer, bile duct cancer, gastrointestinal carcinoidtumors, esophageal cancer, gall bladder cancer, appendix cancer, smallintestine cancer, stomach (gastric) cancer, cancer of the centralnervous system, skin cancer (e.g., melanoma), choriocarcinoma, head andneck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma,neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin'slymphoma, Burkitt's lymphoma, Small Cell lymphoma, Large Cell lymphoma,monocytic leukemia, myelogenous leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, and multiple myeloma.

As used herein the terms “effective amount” and “therapeuticallyeffective amount” refer to a dose of a substance such as animmunoconjugate that produces therapeutic effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11^(th)Edition, 2006, Brunton, Ed., McGraw-Hill; and Remington: The Science andPractice of Pharmacy, 21S^(t) Edition, 2005, Hendrickson, Ed.,Lippincott, Williams & Wilkins).

As used herein, the term “subject” refers to animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In certainembodiments, the subject is a human.

As used herein, the term “administering” refers to parenteral,intravenous, intraperitoneal, intramuscular, intratumoral,intralesional, intranasal or subcutaneous administration, oraladministration, administration as a suppository, topical contact,intrathecal administration, or the implantation of a slow-releasedevice, e.g., a mini-osmotic pump, to the subject.

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding that explicit value. If “X”were the value, “about X” or “around X” would indicate a value from 0.9Xto 1.1X, e.g., from 0.95X to 1.05X or from 0.99X to 1.01X. Any referenceto “about X” or “around X” specifically indicates at least the values X,0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and1.05X. Thus, “about X” and “around X” are intended to teach and providewritten description support for a claim limitation of, e.g., “0.98X.”

Antibody Adjuvant Immunoconjugates

The invention provides immunoconjugates containing an antibody constructcomprising an antigen binding domain and an Fc domain, an adjuvantmoiety, and a linker, wherein each adjuvant moiety is covalently bondedto the antibody via the linker.

Immunoconjugates as described herein can provide an unexpectedlyincreased activation response of an antigen presenting cell (APC). Thisincreased activation can be detected in vitro or in vivo. In some cases,increased APC activation can be detected in the form of a reduced timeto achieve a specified level of APC activation. For example, in an invitro assay, % APC activation can be achieved at an equivalent dose withan immunoconjugate within 1%, 10%, or 50% of the time required toreceive the same or similar percentage of APC activation with a mixtureof unconjugated antibody and TLR agonist. In some cases, animmunoconjugate can activate APCs (e.g., dendritic cells) and/or NKcells in a reduced amount of time. For example, in some cases, anantibody TLR agonist mixture can activate APCs (e.g., dendritic cells)and/or NK cells and/or induce dendritic cell differentiation afterincubation with the mixture for 2, 3, 4, 5, 1-5, 2-5, 3-5, or 4-7 days;while, in contrast immunoconjugates described herein can activate and/orinduce differentiation within 4 hours, 8 hours, 12 hours, 16 hours, or 1day. Alternatively, the increased APC activation can be detected in theform of a reduced concentration of immunoconjugate required to achievean amount (e.g., percent APCs), level (e.g., as measured by a level ofupregulation of a suitable marker), or rate (e.g., as detected by a timeof incubation required to activate) of APC activation.

Immunoconjugates of the present invention must include an Fc region. AsFIGS. 130A-131E illustrate, non-FcR binding proteins do not activatemyeloid cells when conjugated to Compound 1.

In one embodiment, the immunoconjugates of the present invention providemore than a 5% increase in activity compared to the immunoconjugates ofthe prior art (for example, the immunoconjugates disclosed in the '528patent). In another embodiment, the immunoconjugates of the presentinvention provide more than a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, or 70% increase in activity compared to theimmunoconjugates of the prior art. The increase in activity can beassessed by any means known by one of skill in the art and can includemyeloid activation or assessment by cytokine secretion.

In one embodiment, the immunoconjugates of the present invention providean improved drug to adjuvant ratio. In some embodiments, the averagenumber of adjuvant moieties per immunoconjugate ranges from about 1 toabout 10. The desirable drug to adjuvant ratio can be determined by oneof skill in the depending on the desired effect of the treatment. Forexample, a drug to adjuvant ratio of greater than 1.2 may be desired. Inan embodiment, a drug to adjuvant ratio of greater than 0.2, 0.4, 0.6,0.8, 1, 1.2, 1.4, 1.6. 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6,3.8, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 may be desired. In anotherembodiment, a drug to adjuvant ratio of less than 10.0, 9.0, 8.0, 7.0,6.0, 5.0, 4.0, 3.8, 3.6, 3.4, 3.2, 3.0, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8,1.6, 1.4, 1.2, 0.8, 0.6, 0.4 or 0.2 may be desirable. The drug toadjuvant ratio can be assessed by any means known by one of skill in theart.

The immunoconjugates of the invention contain linking moieties thatcovalently bond the adjuvant moieties to the antibodies. In someembodiments, the immunoconjugate has a structure according to Formula I:

or a pharmaceutically acceptable salt thereof, wherein Ab is anantibody; A is an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety;Adj is an adjuvant moiety; and subscript r is an integer from 1 to 10(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In some embodiments, the immunoconjugate has a structure according toFormula Ia:

or a pharmaceutically acceptable salt thereof, wherein

Ab is an antibody;

A is an unmodified amino acid sidechain in the antibody or a modifiedamino acid sidechain in the antibody;

Z is a linking moiety;

R¹ is selected from H and C₁₋₄ alkyl; or

Z, R¹, and the nitrogen atom to which they are attached form a linkingmoiety comprising a 5- to 8-membered heterocycle;

each Y is independently CHR², wherein R² is selected from H, OH, andNH₂,

R³ is selected from C₁₋₆ alkyl and 2- to 6-membered heteroalkyl, each ofwhich is optionally substituted with one or more members selected fromthe group consisting of halo, hydroxy, amino, oxo (═O), alkylamino,amido, acyl, nitro, cyano, and alkoxy;

X is selected from O and CH₂;

-   -   subscript n is an integer from 1 to 12 (i.e., 1, 2, 3, 4, 5, 6,        7, 8, 9, 10, 11, or 12); and subscript r is an integer from 1 to        10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).        In certain embodiments of the immunoconjugate of Formula Ia,        subscript n is an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or        6).

In some embodiments, the immunoconjugate has a structure according toFormula Ib:

or a pharmaceutically acceptable salt thereof, wherein

Ab is an antibody;

A is an unmodified amino acid sidechain in the antibody or a modifiedamino acid sidechain in the antibody;

Z is a linking moiety;

R¹ is selected from H and C₁₋₄ alkyl; or

Z, R¹, and the nitrogen atom to which they are attached form a linkingmoiety comprising a 5- to 8-membered heterocycle;

each Y is independently CHR², wherein R² is selected from H, OH, andNH₂;

X is selected from O and CH₂;

-   -   subscript n is an integer from 1 to 12 (i.e., 1, 2, 3, 4, 5, 6,        7, 8, 9, 10, 11, or 12);    -   and W is selected from the group consisting of O and CH₂.

In some embodiments, the immunoconjugate has a structure according toFormula Ic:

or a pharmaceutically acceptable salt thereof, wherein

Ab is an antibody;

subscript r is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10);

A is an unmodified amino acid sidechain in the antibody or a modifiedamino acid sidechain in the antibody;

Z is a linking moiety; and

R¹ is selected from H and C₁₋₄ alkyl; or

Z, R¹, and the nitrogen atom to which they are attached form a linkingmoiety comprising a 5- to 8-membered heterocycle; and

R² is selected from H, OH, and NH₂.

In some embodiments, the immunoconjugate has a structure according toFormula Id:

or a pharmaceutically acceptable salt thereof, wherein Ab is anantibody; A is an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; R² is selected from H,OH, and NH₂; and subscript r is an integer from 1 to 10 (i.e., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10). In certain embodiments, subscript r is aninteger from 1 to 4 (i.e., 1, 2, 3, or 4). In certain embodiments of theimmunoconjugates of Formula I and Formulae Ia-Id, A is a thiol-modifiedlysine sidechain. In some embodiments of the immunoconjugates of FormulaI and Formulae Ia-Id, A is a cysteine sidechain.

In some embodiments, Z is selected from:

wherein subscript x is an integer from 1 to 12 (i.e., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12); subscript y is an integer from 1 to 30 (i.e.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30); the dashed line (“

”) represents the point of attachment to the adjuvant moiety; and thewavy line (“

”) represents the point of attachment to an amino acid sidechain in theantibody.

In some embodiments, the immunoconjugate has a structure according toFormula II:

or a pharmaceutically acceptable salt thereof, wherein Ab is anantibody; wherein A is an unmodified amino acid sidechain in theantibody or a modified amino acid sidechain in the antibody; wherein Adjis an adjuvant moiety; wherein subscript r is an integer 1 to 10 (i.e.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and wherein:

-   -   Z¹ is selected from —C(O)—, —C(O)NH—, —CH₂—;    -   Z² and Z⁴ are independently selected from a bond, C₁₋₃₀        alkylene, and 3- to 30-membered heteroalkylene, wherein:        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by —C(O)—, —NR^(a)C(O)—, or            —C(O)NR^(a)—,        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            carbocycle,        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            heterocycle having one to four heteroatoms selected from O,            S, and N, and        -   each R^(a) is independently selected from H and C₁₋₆ alkyl;    -   Z³ is selected from a bond, a divalent peptide moiety, and a        divalent polymer moiety; and    -   Z⁵ is bonded to the sidechain of an amino acid sidechain in the        antibody.

In some embodiments, the immunoconjugate has a structure according toFormula IIa:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Z¹ is selected from —C(O)—, —C(O)NH—, —CH₂—;    -   Z² and Z⁴ are independently selected from a bond, C₁₋₃₀        alkylene, and 3- to 30-membered heteroalkylene, wherein:        -   one or more groupings of adjacent atoms in the C₁₋₃₀ alkyl            and 3- to 30-membered heteroalkylene are optionally and            independently replaced by —C(O)—, —NR^(a)C(O)—, or            —C(O)NR^(a)—;        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            carbocycle,        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            heterocycle having one to four heteroatoms selected from O,            S, and N, and        -   each R^(a) is independently selected from H and C₁₋₆ alkyl;    -   Z³ is selected from a bond, a divalent peptide moiety, and a        divalent polymer moiety; and    -   Z⁵ is selected from an amine-bonded moiety and a thiol-bonded        moiety.

In certain embodiments of the immunoconjugates of Formula II and FormulaIIa, Z⁵ is a thiol-bonded moiety. In certain embodiments of theimmunoconjugates of Formula II and Formula IIa, Z⁵ is a thiol-bondedmoiety and A is a thiol-modified lysine sidechain. In certainembodiments of the immunoconjugates of Formula II and Formula IIa, Z⁵ isa thiol-bonded moiety and A is a cysteine sidechain.

In certain embodiments of the immunoconjugates of Formula II and FormulaIIa, the linking moiety (i.e., the structural components between theadjuvant (“Adj”) and the amino acid (“A”)) includes a structure selectedfrom:

wherein Z¹, Z², Z³, and Z⁴ are described as above; the dashed line (“

”) represents the point of attachment to the adjuvant moiety; and thewavy line (“

”) represents the point of attachment to an amino acid sidechain in anantibody.

In some embodiments, Z³ is a divalent peptide moiety. In someembodiments, the peptide includes a first residue selected from analanine residue, a valine residue, a leucine residue, an isoleucineresidue, a methionine residue, a phenylalanine residue, a tryptophanresidue, and a proline residue. In some such embodiments, the peptideincludes a second amino acid selected from an unprotected lysineresidue, a protected lysine residue, an unprotected arginine residue, aprotected arginine residue, a histidine residue, an unprotectedornithine residue, a protected ornithine residue, a lysine residue, anda citrulline. In some embodiments, the peptide includes a first residueselected from a phenylalanine residue and a valine residue. In some suchembodiments, the peptide includes a second residue selected from alysine residue and a citrulline residue. Typically, the peptide moietywill contain about 2-12 amino acid residues. For example, the peptidecan contain from 2 to 8 amino acid residues, or from 2 to 4 amino acidresidues. In some embodiments, the peptide is dipeptide. In someembodiments, the peptide is a tetrapeptide.

In some embodiments, the peptide is selected from Phe-Lys; Val-Lys;Phe-Phe-Lys; D-Phe-Phe-Lys; Gly-Phe-Lys; Ala-Lys; Val-Cit; Val-Ala;Phe-Cit; Leu-Cit; Ile-Cit; Trp-Cit; Phe-Ala; Gly-Phe-Leu-Gly;Ala-Leu-Ala-Leu; Phe-N⁹-tosyl-Arg; and Phe-N⁹-nitro-Arg. In someembodiments, the peptide can be cleaved by a protease such as cathepsinB, cathepsin C, or cathepsin D. Cathepsin B-sensitive peptides can beparticularly useful as linker components, because cathepsin B isimplicated in a number of pathologies and oncogenic processes. Whileexpression and activity of cathepsin B is tightly regulated in healthytissues and organs, regulation can be altered at multiple levels intumors and other malignancies. Overexpression of cathepsin B has beenobserved in various cancers, including brain, lung, prostate, breast,and colorectal cancer. See, e.g., Gondi et al., Expert Opin. Ther.Targets, 2013; 17(3): 281-291. Linkers containing cathepsin B-sensitivepeptide components, such as Phe-Lys and Val-Cit dipeptides, cantherefore be cleaved when an immunoconjugate reaches a malignant targetsuch as a tumor in a subject. Because these peptide components aregenerally insensitive to enzymes in the circulatory system and healthytissues, the adjuvant moieties are not released before theimmunoconjugate reaches the target in the subject.

In some embodiments, Z² is selected from the group consisting C₁₋₃₀alkylene and 3- to 30-membered heteroalkylene, wherein one or moregroupings of adjacent atoms are optionally and independently replaced by—C(O)—, —NHC(O)—, or —C(O)NH—; and one or more groupings of adjacentatoms are optionally and independently replaced by a 4- to 8-membered,divalent carbocycle. In some embodiments, Z² is selected from:

wherein the dashed line (“

”) represents the point of attachment to Z¹, and the wavy line (“

”) represents the point of attachment to Z³.

In certain embodiments, —Z¹—Z²— is:

wherein the dashed line (“

”) represents the point of attachment to the adjuvant moiety and thewavy line (“

”) represents the point of attachment to Z³. In some such embodiments,Z³ is a divalent peptide moiety selected from Phe-Lys and Val-Cit.

In some embodiments, Z⁴ is C₁₋₃₀ alkylene, wherein one or more groupingsof adjacent atoms are optionally and independently replaced by —C(O)—,—NHC(O)—, or —C(O)NH—; and one or more groupings of adjacent atoms areoptionally and independently replaced by a 4- to 8-membered, divalentcarbocycle. In some embodiments, Z⁴ is selected from:

wherein the dashed line (“

”) represents the point of attachment to Z³, and the wavy line (“

”) represents the point of attachment to Z⁵. In some such embodiments,Z³ is a divalent peptide moiety selected from Phe-Lys and Val-Cit.

One of skill in the art will appreciate that the adjuvant moieties inthe conjugates can be covalently bonded to the antibodies using variouschemistries for protein modification, and that the linking moietiesdescribed above result from the reaction of protein functional groups(i.e., amino acid side chains), with reagents having reactive linkergroups. A wide variety of such reagents are known in the art. Examplesof such reagents include, but are not limited to, N-hydroxysuccinimidyl(NHS) esters and N-hydroxysulfosuccinimidyl (sulfo-NHS) esters (aminereactive); carbodiimides (amine and carboxyl reactive); hydroxymethylphosphines (amine reactive); maleimides (thiol reactive); halogenatedacetamides such as N-iodoacetamides (thiol reactive); aryl azides(primary amine reactive); fluorinated aryl azides (reactive viacarbon-hydrogen (C—H) insertion); pentafluorophenyl (PFP) esters (aminereactive); tetrafluorophenyl (TFP) esters (amine reactive); imidoesters(amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones(thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiolreactive); and benzophenone derivatives (reactive via C—H bondinsertion). Further reagents include but are not limited to thosedescribed in Hermanson, Bioconjugate Techniques 2nd Edition, AcademicPress, 2008.

Linkers containing maleimide groups, vinyl sulfone groups, pyridyldisulfide groups, and halogenated acetamide groups are particularlyuseful for covalent bonding to thiol groups in an antibody. Thiol groupsin an antibody are generally located in cysteine sidechains. Free thiolgroups may be present in naturally-occurring, solvent-accessiblecysteine residues in the antibody. Free thiols can also be present inengineered cysteine residues, as described below. In addition, thiolgroups can be generated via full or partial reduction of disulfidelinkages between cysteine sidechains in an antibody. Thiol groups can bealso appended to lysine sidechains using known methods with reagentsincluding, but not limited to, 2-iminothiolane (Traut's reagent),N-succinimidyl-S-acetylthioacetate (SATA), and SATP(N-succinimidyl-S-acetylthiopropionate). When the antibody is modifiedwith acetylated reagents like SATA and SATP, acetyl groups can beremoved via hydrolysis with hydroxylamine or similar reagents in orderto generate free thiol groups for further conjugation. See, e.g., Trautet al. (Biochem., 12(17): 3266-3273 (1973)) and Duncan et al. (Anal.Biochem., 132(1): 68-73 (1983)).

The linker can have any suitable length such that when the linker iscovalently bound to the antibody construct and the adjuvant moiety, thefunction of the antibody construct and the adjuvant moiety ismaintained. The linker can have a length of about 3 Å or more, forexample, about 4 Å or more, about 5 Å or more, about 6 Å or more, about7 Å or more, about 8 Å or more, about 9 Å or more, or about 10 Å ormore. Alternatively, or in addition to, the linker can have a length ofabout 50 Å or less, for example, about 45 Å or less, about 40 Å or less,about 35 Å or less, about 30 Å or less, about 25 Å or less, about 20 Åor less, or about 15 Å or less. Thus, the linker can have a lengthbounded by any two of the aforementioned endpoints. The linker can havea length from about 3 Å to about 50 Å, for example, from about 3 Å toabout 45 Å, from about 3 Å to about 40 Å, from about 3 Å to about 35 Å,from about 3 Å to about 30 Å, from about 3 Å to about 25 Å, from about 3Å to about 20 Å, from about 3 Å to about 15 Å, from about 5 Å to about50 Å, from about 5 Å to about 25 Å, from about 5 Å to about 20 Å, fromabout 10 Å to about 50 Å, from about 10 Å to about 20 Å, from about 5 Åto about 30 Å, or from about 5 Å to about 15 Å. In preferredembodiments, the linker has a length from about 3 Å to about 20 Å.

Accordingly, the invention provides embodiments wherein the adjuvantmoieties are covalently bonded to the antibody using a reagent (orcovalent bonding reagent (“CBR”)) selected from:

wherein X is halogen (e.g., iodo, bromo, or chloro); R′ is H or sulfo;R″ is optionally substituted aryl (e.g., 3-carboxy-4-nitrophenyl) oroptionally substituted heteroaryl (e.g., pyridin-2-yl); R′″ isoptionally substituted alkyl (e.g., methoxy); Z¹, Z², Z³, and Z⁴ are asdescribed above; and the dashed line (“

”) represents the point of attachment to the adjuvant moiety.

In some embodiments, the linker moiety —Z¹-Z²-Z³-Z⁴-Z⁵— is:

wherein the dashed line (“

”) represents the point of attachment to the adjuvant moiety, and thewavy line (“

”) represents the point of attachment to an amino acid sidechain theantibody. In some such embodiments, the amino acid sidechain is acysteine sidechain or a modified lysine sidechain containing a thiolgroup.

In some embodiments, the immunoconjugate has a structure according toFormula III:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain, Adj is an adjuvant, G is CH₂, C═O,or a bond, L is a linker, and subscript r is an integer from 1 to 10(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In certain embodiments of theimmunoconjugate of Formula III, that antibody does not contain athiol-modified lysine sidechain.

In some embodiments, L is selected from:

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; a is an integerfrom 1 to 40; each A is independently selected from any amino acid;subscript c is an integer from 1 to 20; the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to

In some embodiments, the immunoconjugate has a structure according toFormula IIIa:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; subscript a is aninteger from 1 to 40; and subscript r is an integer from 1 to 10 (i.e.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In some embodiments, the immunoconjugate has a structure according toFormula IIIb:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; subscript a is an integer from 1 to 40; and subscript r is aninteger from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In some embodiments, the immunoconjugate has a structure according toFormula

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; each A isindependently selected from any amino acid; subscript c is an integerfrom 1 to 20; and subscript r is an integer from 1 to 10 (i.e., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10).

In some embodiments, the immunoconjugate has a structure according toFormula IIId:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; subscript c is aninteger from 1 to 20; and subscript r is an integer from 1 to 10 (i.e.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In some embodiments, the immunoconjugate has a structure according toFormula IIIe:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; and subscript r isan integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In some embodiments, the immunoconjugate has a structure according toFormula IIIf:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; and subscript r isan integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In some embodiments, the immunoconjugate has a structure according toFormula IIIg:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; and subscript r isan integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

Accordingly, the immunoconjugate can have a structure according toFormula IVa-Formula IVk:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; and subscript ris an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Incertain embodiments, subscript r is an integer from 1 to 4 (i.e., 1, 2,3, or 4).

In certain embodiments, the immunoconjugate has a structure selectedfrom:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain and subscript r is an integer from 1to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In certain embodiments,subscript r is an integer from 1 to 4 (i.e., 1, 2, 3, or 4).

In certain embodiments, the immunoconjugate has a structure selectedfrom:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain and subscript r is an integer from 1to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In certain embodiments,subscript r is an integer from 1 to 4 (i.e., 1, 2, 3, or 4).

In a second aspect, the invention provides an improved method forproducing an immunoconjugate of Formula III from one or more compoundsof Formula V and an antibody of Formula VI, the method comprising thestep of:

wherein Adj is an adjuvant; G is CH₂, C═O, or a bond; L is a linker; Eis an ester; Formula VI is an antibody with at least one lysine sidechain; and subscript r is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10). In certain embodiments, the adjuvant (“Adj”) is aTLR agonist.

Any suitable linker can be used provided it can be bound to the antibody(compound of Formula VI) through an ester. For example, the linker (“L”)can have the following formula

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or8) carbon units; subscript a is an integer from 1 to 40; the dashed line(“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to E. In some embodiments,subscript a is an integer from 1 to 20. In some embodiments, subscript ais an integer from 1 to 10. In some embodiments, subscript a is aninteger from 1 to 5. In some embodiments, subscript a is an integer from1 to 3. In certain embodiments, R is present and is a linear orbranched, cyclic or straight, saturated or unsaturated alkyl,heteroalkyl, aryl, or heteroaryl chain comprising from 1 to 8 (i.e., 1,2, 3, 4, 5, 6, 7, or 8) carbon units.

The linker (“L”) can have the following formula

wherein subscript a is an integer from 1 to 40; the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to E.

In some embodiments, subscript a is an integer from 1 to 20. In someembodiments, subscript a is an integer from 1 to 10. In someembodiments, subscript a is an integer from 1 to 5. In some embodiments,subscript a is an integer from 1 to 3.

The linker (“L”) can also have the following formula

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or8) carbon units; each A is independently selected from any amino acid;subscript c is an integer from 1 to 20; the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to E. In some embodiments,subscript c is an integer from 1 to 10. In some embodiments, subscript cis an integer from 1 to 5. In some embodiments, subscript c is aninteger from 1 to 2. In certain embodiments, R is present and is alinear or branched, cyclic or straight, saturated or unsaturated alkyl,heteroalkyl, aryl, or heteroaryl chain comprising from 1 to 8 (i.e., 1,2, 3, 4, 5, 6, 7, or 8) carbon units.

The linker (“L”) can also have the following formula

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or8) carbon units; subscript c is an integer from 1 to 20; the dashed line(“

”) represents the point of attachment td

and the wavy line (“

”) represents the point of attachment to E. In some embodiments,subscript c is an integer from 1 to 10. In some embodiments, c is aninteger from 1 to 5. In certain embodiments, R is present and is alinear or branched, cyclic or straight, saturated or unsaturated alkyl,heteroalkyl, aryl, or heteroaryl chain comprising from 1 to 8 (i.e., 1,2, 3, 4, 5, 6, 7, or 8) carbon units.

The linker (“L”) can also have the following formula

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or8) carbon units; the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to E. In certain embodiments, R ispresent and is a linear or branched, cyclic or straight, saturated orunsaturated alkyl, heteroalkyl, aryl, or heteroaryl chain comprisingfrom 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units.

The linker (“L”) can also have the following formula

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or8) carbon units; the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to E. In certain embodiments, R ispresent and is a linear or branched, cyclic or straight, saturated orunsaturated alkyl, heteroalkyl, aryl, or heteroaryl chain comprisingfrom 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units.

The linker (“L”) can also have the following formula

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or8) carbon units; the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to E. In certain embodiments, R ispresent and is a linear or branched, cyclic or straight, saturated orunsaturated alkyl, heteroalkyl, aryl, or heteroaryl chain comprisingfrom 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units.

In some embodiments, the compound of Formula V is selected from:

wherein G is CH₂, C═O, or a bond; R is optionally present and is alinear or branched, cyclic or straight, saturated or unsaturated alkyl,heteroalkyl, aryl, or heteroaryl chain comprising from 1 to 8 carbonunits; subscript a is an integer from 1 to 40; each A is independentlyselected from any amino acid; subscript c is an integer from 1 to 20,and E is an ester.

As previously discussed, there are many ways of forming animmunoconjugate. Each of the prior art methods suffers from downsides.The present method includes a one-step process which conjugates anadjuvant, modified to include a linker, to the lysine side chain of anantibody (compound of Formula VI). This process is possible by using anester. The ester can be any suitable ester capable of linking thecompound of Formula V to a lysine side chain of an antibody (compound ofFormula VI).

For example, the ester of Formula V can be an N-hydroxysuccinimide(“NHS”) ester of the formula:

wherein the wavy line (“

”) represents the point of attachment to the linker (“L”).

The ester of Formula V can also be a sulfo-N-hydroxysuccinimide ester ofthe formula:

wherein M is any cation and the wavy line (“

”) represents the point of attachment to the linker (“L”). For example,the cation counter ion (“M”) can be a proton, ammonium, a quaternaryamine, a cation of an alkali metal, a cation of an alkaline earth metal,a cation of a transition metal, a cation of a rare-earth metal, a maingroup element cation, or a combination thereof.

The ester of Formula V can also be a phenol ester of the formula:

wherein each R₂ is independently selected from hydrogen or fluorine andthe wavy line (“

”) represents the point of attachment to the linker (“L”).

The ester of Formula V can also be a phenol ester of the formula:

wherein the wavy line (“

”) represents the point of attachment to the linker (“L”).

In some embodiments, the antibody of Formula VI and the ester of FormulaV are combined in any suitable aqueous buffer. An exemplary list ofsuitable aqueous buffers is phosphate buffered saline, borate bufferedsaline, and tris buffered saline.

Using a tetrafluorophenyl (“TFP”) or pentafluorophenyl (“PFP”) isespecially effective in synthesizing the immunoconjugates of the presentinvention.

Accordingly, an exemplary, but non-limiting, list of compounds ofFormula V is:

wherein Adj is an adjuvant and M is any cation. For example, the cationcounter ion (“M”) can be a proton, ammonium, a quaternary amine, acation of an alkali metal, a cation of an alkaline earth metal, a cationof a transition metal, a cation of a rare-earth metal, a main groupelement cation, or a combination thereof.

Accordingly, the one or more compounds of Formula V and an antibody ofFormula VI can be combined to form an immunoconjugate of Formula III. Anexemplary, but non-limiting list of immunoconjugates of Formula III is:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; subscript a is aninteger from 1 to 40; each A is independently selected from any aminoacid; subscript c is an integer from 1 to 20; and subscript r is aninteger from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In a third aspect, the invention provides an immunoconjugate containlinking moieties that covalently bond the adjuvant moieties, comprisingan oligonucleotide, to the antibodies. In certain embodiments, theimmunoconjugate is an A-type CPG oligonucleotide immunoconjugateselected from an immunoconjugate of Formula VIIa:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodybound at an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety;subscript r is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10); lowercase nucleotides signify a phosphorothioate linkage; anduppercase nucleotides signify a phosphodiester linkage. In certainembodiments, the linking moiety (“Z”) is as defined above and herein.

In certain embodiments, the immunoconjugate is an B-type CPGoligonucleotide immunoconjugate selected from an immunoconjugate ofFormula VIIb:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodybound at an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety;subscript r is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10); and uppercase nucleotides signify a phosphorothioate linkage. Incertain embodiments, the linking moiety (“Z”) is as defined above andherein.

In certain embodiments, the immunoconjugate is an C-type CPGoligonucleotide immunoconjugate selected from an immunoconjugate ofFormula VIIc:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodybound at an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety;subscript r is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10); and uppercase nucleotides signify a phosphorothioate linkage. Incertain embodiments, the linking moiety (“Z”) is as defined above andherein.

In certain embodiments, the immunoconjugate is an PolyI:Coligonucleotide immunoconjugate selected from an immunoconjugate ofFormula VIId:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodybound at an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety;and subscript r is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10). In certain embodiments, the linking moiety (“Z”) is asdefined above and herein.

Adjuvants

In some embodiments, the adjuvant moiety is a compound that elicits animmune response. In some embodiments, the adjuvant moiety is a patternrecognition receptor (“PRR”) agonist. Any adjuvant capable of activatinga pattern recognition receptor (PRR) can be installed in theimmunoconjugates of the invention. As used herein, the terms “Patternrecognition receptor” and “PRR” refer to any member of a class ofconserved mammalian proteins which recognize pathogen-associatedmolecular patterns (“PAMPs”) or damage-associated molecular patterns(“DAMPs”), and act as key signaling elements in innate immunity. Patternrecognition receptors are divided into membrane-bound PRRs, cytoplasmicPRRs, and secreted PRRs. Examples of membrane-bound PRRs includeToll-like receptors (“TLRs”) and C-type lectin receptors (“CLRs”).Examples of cytoplasmic PRRs include NOD-like receptors (“NLRs”) andRig-I-like receptors (“RLRs”). In some embodiments, the immunoconjugatecan have more than one distinct PRR adjuvant moiety.

In certain embodiments, the adjuvant moiety in an immunoconjugate of theinvention is a Toll-like receptor (TLR) agonist. Suitable TLR agonistsinclude TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11, or any combination thereof (e.g., TLR7/8 agonists). Any adjuvantcapable of activating a Toll-like receptor (TLR) can be installed in theimmunoconjugates of the invention. Toll-like receptors (TLRs) are type-Itransmembrane proteins that are responsible for initiation of innateimmune responses in vertebrates. TLRs recognize a variety ofpathogen-associated molecular patterns from bacteria, viruses, and fungiand act as a first line of defense against invading pathogens. TLRselicit overlapping yet distinct biological responses due to differencesin cellular expression and in the signaling pathways that they initiate.Once engaged (e.g., by a natural stimulus or a synthetic TLR agonist)TLRs initiate a signal transduction cascade leading to activation ofNF-κB via the adapter protein myeloid differentiation primary responsegene 88 (MyD88) and recruitment of the IL-1 receptor associated kinase(IRAK). Phosphorylation of IRAK then leads to recruitment ofTNF-receptor associated factor 6 (TRAF6), which results in thephosphorylation of the NF-κB inhibitor I-κB. As a result, NF-κB entersthe cell nucleus and initiates transcription of genes whose promoterscontain NF-κB binding sites, such as cytokines. Additional modes ofregulation for TLR signaling include TIR-domain containingadapter-inducing interferon-β (TRIF)-dependent induction of TRAF6 andactivation of MyD88 independent pathways via TRIF and TRAF3, leading tothe phosphorylation of interferon response factor three (IRF3).Similarly, the MyD88 dependent pathway also activates several IRF familymembers, including IRF5 and IRF7 whereas the TRIF dependent pathway alsoactivates the NF-κB pathway.

Examples of TLR3 agonists include Polyinosine-polycytidylic acid (poly(I:C)), Polyadenylic-polyuridylic acid (poly (A:U), andpoly(I)-poly(C12U).

Examples of TLR4 agonists include Lipopolysaccharide (LPS) andMonophosphoryl lipid A (MPLA).

An example of a TLR5 agonist includes Flagellin.

Examples of TLR9 agonists include single strand CpGoligodeoxynucleotides (CpG ODN). Three major classes of stimulatory CpGODNs have been identified based on structural characteristics andactivity on human peripheral blood mononuclear cells (PBMCs), inparticular B cells and plasmacytoid dendritic cells (pDCs). These threeclasses are Class A (Type D), Class B (Type K) and Class C.

Examples of Nod Like Receptor (NLR) agonists include acylated derivativeof iE-DAP, D-gamma-Glu-mDAP, L-Ala-gamma-D-Glu-mDAP, Muramyldipeptidewith a C18 fatty acid chain, Muramyldipeptide, muramyl tripeptide, andN-glycolylated muramyldipeptide.

Examples of RIG-I-Like receptor (RLR) agonists include 5′ppp-dsrna(5′-pppGCAUGCGACCUCUGUUUGA-3′ (SEQ ID NO: 3): 3′-CGUACGCUGGAGACAAACU-5′(SEQ ID NO: 4)), and Poly(deoxyadenylic-deoxythymidylic) acid(Poly(dA:dT))

Additional immune-stimulatory compounds, such as cytosolic DNA andunique bacterial nucleic acids called cyclic dinucleotides, can berecognized by stimulator of interferon genes (“STING”), which can act acytosolic DNA sensor. ADU-S1OO can be a STING agonist. Non-limitingexamples of STING agonists include: Cyclic [G(2′,5′)pA(2′,5′)p](2′2′-cGAMP), cyclic [G(2′,5′)pA(3′,5′)p] (2′3′-cGAMP), cyclic[G(3′,5′)pA(3′,5′)p] (3′3′-cGAMP), Cyclic di-adenylate monophosphate(c-di-AMP), 2′,5′-3′,5′-c-diAMP (2′3′-c-di-AMP), Cyclic di-guanylatemonophosphate (c-di-GMP), 2′,5′-3′,5′-c-diGMP (2′3′-c-di-GMP), Cyclicdi-inosine monophosphate (c-di-IMP), Cyclic di-uridine monophosphate(c-di-UMP), KIN700, KIN1148, KIN600, KIN500, KIN1OO, KIN101, KIN400,KIN2000, or SB-9200 can be recognized.

Any adjuvant capable of activating TLR7 and/or TLR8 can be installed inthe immunoconjugates of the invention. Examples of TLR7 agonists andTLR8 agonists are described, e.g., by Vacchelli et al. (OncoImmunology,2: 8, e25238, DOI: 10.4161/onci.25238 (2013)) and Carson et al. (U.S.Patent Application Publication 2013/0165455, which is herebyincorporated by reference in its entirety). TLR7 and TLR8 are bothexpressed in monocytes and dendritic cells. In humans, TLR7 is alsoexpressed in plasmacytoid dendritic cells (pDCs) and B cells. TLR8 isexpressed mostly in cells of myeloid origin, i.e., monocytes,granulocytes, and myeloid dendritic cells. TLR7 and TLR8 are capable ofdetecting the presence of “foreign” single-stranded RNA within a cell,as a means to respond to viral invasion. Treatment of TLR8-expressingcells, with TLR8 agonists can result in production of high levels ofIL-12, IFN-γ, IL-1, TNF-u, IL-6, and other inflammatory cytokines.Similarly, stimulation of TLR7-expressing cells, such as pDCs, with TLR7agonists can result in production of high levels of IFN-α and otherinflammatory cytokines. TLR7/TLR8 engagement and resulting cytokineproduction can activate dendritic cells and other antigen-presentingcells, driving diverse innate and acquired immune response mechanismsleading to tumor destruction.

Examples of TLR7, TLR8 or TLR7/8 agonists include but are not limitedto: Gardiquimod(1-(4-amino-2-ethylaminomethylimidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol),Imiquimod (R837) (agonist for TLR7), loxoribine (agonist for TLR7), IRM1(1-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo-[4,5-c]quinolin-4-amine),IRM2(2-methyl-l-[2-(3-pyridin-3-ylpropoxy)ethyl]-1H-imidazo[4,5-c]quinolin-4-amine)(agonist for TLR8), IRM3(N-(2-[2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy]ethyl)-N-methylcyclohexanecarboxamide)(agonist for TLR8), CL097(2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine) (agonist forTLR7/8), CL307 (agonist for TLR7), CL264 (agonist for TLR7), Resiquimod(agonist for TLR7/8), 3M-052/MEDI9197 (agonist for TLR7/8), SD-101(N-[(4S)-2,5-dioxo-4-imidazolidinyl]-urea) (agonist for TLR7/8),motolimod(2-amino-N,N-dipropyl-8-[4-(pyrrolidine-1-carbonyl)phenyl]-3H-1-benzazepine-4-carboxamide)(agonist for TLR8), CL075 (3M002,2-propylthiazolo[4,5-c]quinolin-4-amine) (agonist for TLR7/8), andTL8-506 (3H-1-benzazepine-4-carboxylic acid, 2-amino-8-(3-cyanophenyl)-,ethyl ester) (agonist for TLR8).

Examples of TLR2 agonists include but are not limited to an agentcomprisingN-a-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine,palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl) (“Pam3Cys”), e.g.,Pam3Cys, Pam3Cys-Ser-(Lys)4 (also known as “Pam3Cys-SKKKK” and“Pam3CSK4”), Triacyl lipid A (“OM-174”), Lipoteichoic acid (“LTA”),peptidoglycan, and CL419(S-(2,3-bis(palmitoyloxy)-(2RS)propyl)-(R)-cysteinyl spermine).

An example of a TLR2/6 agonist is Pam₂CSK₄(S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysinex 3 CF3COOH).

Examples of TLR2/7 agonist include CL572 (S-(2-myristoyloxyethyl)-(R)-cysteinyl4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl) aniline),CL413(S-(2,3-bis(palmitoyloxy)-(2RS)propyl)-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysyl4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)aniline), andCL401 (S-(2,3-bis(palmitoyloxy)-(2RS)propyl)-(R)-cysteinyl4-((6-amino-2(butyl amino)-8-hydroxy-9H-purin-9-yl)methyl) aniline).

FIGS. 22A-22X shows where TLR agonists CL264, CL401, CL413, CL419,CL553, CL572, Pam₃CSK₄, and Pam₂CSK₄ could be linked to immunoconjugatesof the present invention while maintaining their adjuvant activity.Specifically, the location where the linker should be attached to theadjuvant is circled.

In some embodiments, the adjuvant moiety is an imidazoquinolinecompound. Examples of useful imidazoquinoline compounds include thosedescribed in U.S. Pat. Nos. 5,389,640; 6,069,149; and 7,968,562, whichare hereby incorporated by reference in their entirety.

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein each J independently is hydrogen, OR⁴, or R⁴; each R⁴independently is hydrogen, or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units; Qis optionally present and is an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units;and the dashed line (“

”) represents the point of attachment of the adjuvant. In certainembodiments, Q is present. In certain embodiments, the adjuvant (“Adj”)is of formula:

wherein each R⁴ independently is selected from the group consisting ofhydrogen, or alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl group comprising from 1 to 8(i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units and the dashed line (“

”) represents the point of attachment of the adjuvant.

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein J is hydrogen, OR⁴, or R⁴; each R⁴ independently is hydrogen, oralkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,arylalkyl, and heteroarylalkyl group comprising from 1 to 8 (i.e., 1, 2,3, 4, 5, 6, 7, or 8) carbon units; Q is selected from the groupconsisting of alkyl, or heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl group comprising from 1 to 8(i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units; and the dashed line (“

”) represents the point of attachment of the adjuvant. In certainembodiments, the adjuvant (“Adj”) is of formula:

wherein each R⁴ independently is selected from the group consisting ofhydrogen, or alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl group comprising from 1 to 8(i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units and the dashed line (“

”) represents the point of attachment of the adjuvant.

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein each R⁴ independently is hydrogen, or alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, orheteroarylalkyl group comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7,or 8) carbon units; Q is alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units;and the dashed line (“

”) represents the point of attachment of the adjuvant.

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein each J independently is hydrogen, OR⁴, or R⁴; each R⁴independently is hydrogen, or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units;each U independently is CH or N wherein at least one U is N; eachsubscript t independently is an integer from 1 to 3 (i.e., 1, 2, or 3);Q is optionally present and is an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units;and the dashed line (“

”) represents the point of attachment of the adjuvant. In certainembodiments, Q is present. In certain embodiments, the adjuvant (“Adj”)is of formula:

wherein R⁴ is selected from the group consisting of hydrogen, or alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl,and heteroarylalkyl group comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5,6, 7, or 8) carbon units Q is an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units;and the dashed line (“

”) represents the point of attachment of the adjuvant.In some embodiments, the adjuvant (“Adj”) is of formula:

wherein J is hydrogen, OR⁴, or R⁴; each R⁴ independently is hydrogen, oran alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,arylalkyl, or heteroarylalkyl group comprising from 1 to 8 (i.e., 1, 2,3, 4, 5, 6, 7, or 8) carbon units; R⁵ is hydrogen, or an alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl,or heteroarylalkyl group comprising from 1 to 10 (i.e., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10) carbon units; Q is an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) carbon units;and the dashed line (“

”) represents the point of attachment of the adjuvant. In certainembodiments, the adjuvant (“Adj”) is of formula:

wherein J is hydrogen, OR⁴, or R⁴; each R⁴ independently is selectedfrom the group consisting of hydrogen, or alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, andheteroarylalkyl group comprising from 1 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7,or 8) carbon units; U is CH or N; V is CH₂, O, or NH; each subscript tindependently is an integer from 1 to 3 (i.e., 1, 2, or 3); and thedashed line (“

”) represents the point of attachment of the adjuvant.

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein R¹ is selected from H and C₁₋₄ alkyl; R³ is selected from C₁₋₆alkyl and 2- to 6-membered heteroalkyl, each of which is optionallysubstituted with one or more members selected from the group consistingof halo, hydroxy, amino, oxo (═O), alkylamino, amido, acyl, nitro,cyano, and alkoxy; X is selected from O and CH₂; each Y is independentlyCHR², wherein R² is selected from H, OH, and NH₂, subscript n is aninteger from 1 to 12 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12);and the dashed line (“

”) represents the point of attachment of the adjuvant. Alternatively, R¹and the nitrogen atom to which it is attached can form a linking moietycomprising a 5- to 8-membered heterocycle. In some embodiments,subscript n is an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6). Incertain embodiments, subscript n is an integer from 1 to 3 (i.e., 1, 2,or 3).

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein W is selected from the group consisting of O and CH2; R¹ isselected from H and C₁₋₄ alkyl; each Y is independently CHR², wherein R²is selected from H, OH, and NH₂; subscript n is an integer from 1 to 12(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); and the dashed line (“

”) represents the point of attachment of the adjuvant. Alternatively, R¹and the nitrogen atom to which it is attached can form a linking moietycomprising a 5- to 8-membered heterocycle. In some embodiments,subscript n is an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6). Incertain embodiments, subscript n is an integer from 1 to 3 (i.e., 1, 2,or 3).

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein W is selected from the group consisting of O and CH₂; R¹ isselected from H and C₁₋₄ alkyl; each Y is independently CHR², wherein R²is selected from H, OH, and NH₂; subscript n is an integer from 1 to 12(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); and the dashed line (“

”) represents the point of attachment of the adjuvant. Alternatively, R¹and the nitrogen atom to which it is attached can form a linking moietycomprising a 5- to 8-membered heterocycle. In some embodiments,subscript n is an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6). Incertain embodiments, subscript n is an integer from 1 to 3 (i.e., 1, 2,or 3).

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein W is selected from the group consisting of O and CH₂; X isselected from O and CH₂; each Y is independently CHR², wherein R² isselected from H, OH, and NH₂; subscript n is an integer from 1 to 12(i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12); and the dashed line (“

”) represents the point of attachment of the adjuvant. In someembodiments, subscript n is an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5,or 6). In certain embodiments, subscript n is an integer from 1 to 3(i.e., 1, 2, or 3).

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein R¹ is selected from H and C₁₋₄ alkyl; R² is selected from H, OH,and NH₂; and the dashed line (“

”) represents the point of attachment of the adjuvant.

In some embodiments, the adjuvant (“Adj”) is of formula:

wherein R¹ is selected from H and C₁₋₄ alkyl; R² is selected from H, OH,and NH₂; and the dashed line (“

”) represents the point of attachment of the adjuvant.

In certain embodiments, the adjuvant (“Adj”) is:

wherein the dashed line (“

”) represents the point of attachment of the adjuvant.

In some embodiments, the adjuvant is not a fluorophore. In someembodiments, the adjuvant is not a radiodiagnostic compound. In someembodiments, the adjuvant is not a radiotherapeutic compound. In someembodiments, the adjuvant is not a tubulin inhibitor. In someembodiments, the adjuvant is not a DNA crosslinker/alkylator. In someembodiments, the adjuvant is not a topoisomerase inhibitor.

Antibodies

The antibodies in the immunoconjugates can be allogeneic antibodies. Theterms “allogeneic antibody” or “alloantibody” refer to an antibody thatis not from the individual in question (e.g., an individual with a tumorand seeking treatment), but is from the same species, or is from adifferent species, but has been engineered to reduce, mitigate, or avoidrecognition as a xeno-antibody (e.g., non-self). For example, the“allogeneic antibody” can be a humanized antibody. Unless specificallystated otherwise, “antibody” and “allogeneic antibodies” as used hereinrefer to immunoglobulin G (IgG) or immunoglobulin A (IgA).

If a cancer cell of a human individual is contacted with an antibodythat was not generated by that same person (e.g., the antibody wasgenerated by a second human individual, the antibody was generated byanother species such as a mouse, the antibody is a humanized antibodythat was generated by another species, etc.), then the antibody isconsidered to be allogeneic (relative to the first individual). Ahumanized mouse monoclonal antibody that recognizes a human antigen(e.g., a cancer-specific antigen, an antigen that is enriched in and/oron cancer cells, etc.) is considered to be an “alloantibody” (anallogeneic antibody).

In some embodiments, the antibody is a polyclonal allogeneic IgGantibody. In some embodiments, the antibody is present in a mixture ofpolyclonal IgG antibodies with a plurality of binding specificities. Insome cases, the antibodies of the mixture specifically bind to differenttarget molecules, and in some cases the antibodies of the mixturespecifically bind to different epitopes of the same target molecule.Thus, a mixture of antibodies can in some cases include more than oneimmunoconjugate of the invention (e.g., adjuvant moieties can becovalently bonded to antibodies of a mixture, e.g., a mixture ofpolyclonal IgG antibodies, resulting in a mixture of antibody-adjuvantconjugates of the invention). A mixture of antibodies can be pooled from2 or more individuals (e.g., 3 or more individuals, 4 or moreindividuals, 5 or more individuals, 6 or more individuals, 7 or moreindividuals, 8 or more individuals, 9 or more individuals, 10 or moreindividuals, etc.). In some cases, pooled serum is used as a source ofalloantibody, where the serum can come from any number of individuals,none of whom are the first individual (e.g., the serum can be pooledfrom 2 or more individuals, 3 or more individuals, 4 or moreindividuals, 5 or more individuals, 6 or more individuals, 7 or moreindividuals, 8 or more individuals, 9 or more individuals, 10 or moreindividuals, etc.). In some cases, the antibodies are isolated orpurified from serum prior to use. The purification can be conductedbefore or after pooling the antibodies from different individuals.

In some cases where the antibodies in the immunoconjugates comprise IgGsfrom serum, the target antigens for some (e.g., greater than 0% but lessthan 50%), half, most (greater than 50% but less than 100%), or even allof the antibodies (i.e., IgGs from the serum) will be unknown. However,the chances are high that at least one antibody in the mixture willrecognize the target antigen of interest because such a mixture containsa wide variety of antibodies specific for a wide variety of targetantigens.

In some embodiments, the antibody is a polyclonal allogeneic IgAantibody. In some embodiments, the antibody is present in a mixture ofpolyclonal IgA antibodies with a plurality of binding specificities. Insome cases, the antibodies of the mixture specifically bind to differenttarget molecules, and in some cases the antibodies of the mixturespecifically bind to different epitopes of the same target molecule.Thus, a mixture of antibodies can in some cases include more than oneimmunoconjugate of the invention (e.g., adjuvant moieties can becovalently bonded to antibodies of a mixture, e.g., a mixture ofpolyclonal IgA antibodies, resulting in a mixture of antibody-adjuvantconjugates of the invention). A mixture of antibodies can be pooled from2 or more individuals (e.g., 3 or more individuals, 4 or moreindividuals, 5 or more individuals, 6 or more individuals, 7 or moreindividuals, 8 or more individuals, 9 or more individuals, 10 or moreindividuals, etc.). In some cases, pooled serum is used as a source ofalloantibody, where the serum can come from any number of individuals,none of whom are the first individual (e.g., the serum can be pooledfrom 2 or more individuals, 3 or more individuals, 4 or moreindividuals, 5 or more individuals, 6 or more individuals, 7 or moreindividuals, 8 or more individuals, 9 or more individuals, 10 or moreindividuals, etc.). In some cases, the antibodies are isolated orpurified from serum prior to use. The purification can be conductedbefore or after pooling the antibodies from different individuals.

In some cases where the antibodies in the immunoconjugates comprise IgAsfrom serum, the target antigens for some (e.g., greater than 0% but lessthan 50%), half, most (greater than 50% but less than 100%), or even allof the antibodies (i.e., IgAs from the serum) will be unknown. However,the chances are high that at least one antibody in the mixture willrecognize the target antigen of interest because such a mixture containsa wide variety of antibodies specific for a wide variety of targetantigens.

In some cases, the antibody in the immunoconjugates includes intravenousimmunoglobulin (IVIG) and/or antibodies from (e.g., enriched from,purified from, e.g., affinity purified from) IVIG. IVIG is a bloodproduct that contains IgG (immunoglobulin G) pooled from the plasma(e.g., in some cases without any other proteins) from many (e.g.,sometimes over 1,000 to 60,000) normal and healthy blood donors. IVIG iscommercially available. IVIG contains a high percentage of native humanmonomeric IVIG, and has low IgA content. When administeredintravenously, IVIG ameliorates several disease conditions. Therefore,the United States Food and Drug Administration (FDA) has approved theuse of IVIG for a number of diseases including (1) Kawasaki disease; (2)immune-mediated thrombocytopenia; (3) primary immunodeficiencies; (4)hematopoietic stem cell transplantation (for those older than 20 years);(5) chronic B-cell lymphocytic leukemia; and (6) pediatric HIV type 1infection. In 2004, the FDA approved the Cedars-Sinai IVIG Protocol forkidney transplant recipients so that such recipients could accept aliving donor kidney from any healthy donor, regardless of blood type(ABO incompatible) or tissue match. These and other aspects of IVIG aredescribed, for example, in US Patent Application Publications2010/0150942; 2004/0101909; 2013/0177574; 2013/0108619; and2013/0011388; which are hereby incorporated by reference in theirentirety.

In some cases, the antibody is a monoclonal antibody of a definedsub-class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, or IgA₂). If combinationsof antibodies are used, the antibodies can be from the same subclass orfrom different subclasses. For example, the antibodies can be IgG1antibodies. Various combinations of different subclasses, in differentrelative proportions, can be obtained by those of skill in the art. Insome cases, a specific subclass, or a specific combination of differentsubclasses can be particularly effective at cancer treatment or tumorsize reduction. Accordingly, some embodiments of the invention provideimmunoconjugates wherein the antibody is a monoclonal antibody. In someembodiments, the monoclonal antibody is humanized.

In some embodiments, the antibody binds to an antigen of a cancer cell.For example, the antibody can bind to a target antigen that is presentat an amount of at least 10; 100; 1,000; 10,000; 100,000; 1,000,000;2.5×10⁶; 5×10⁶; or 1×10⁷ copies or more on the surface of a cancer cell.

In some embodiments, the antibody binds to an antigen on a cancer orimmune cell at a higher affinity than a corresponding antigen on anon-cancer cell. For example, the antibody may preferentially recognizean antigen containing a polymorphism that is found on a cancer or immunecell as compared to recognition of a corresponding wild-type antigen onthe non-cancer or non-immune cell. In some cases, the antibody binds acancer or immune cell with greater avidity than a non-cancer ornon-immune cell. For example, the cancer or immune cell can express ahigher density of an antigen, thus providing for a higher affinitybinding of a multivalent antibody to the cancer or immune cell.

In some cases, the antibody does not significantly bind non-cancerantigens (e.g., the antibody binds one or more non-cancer antigens withat least 10; 100; 1,000; 10,000; 100,000; or 1,000,000-fold loweraffinity (higher Kd) than the target cancer antigen). In some cases, thetarget cancer antigen to which the antibody binds is enriched on thecancer cell. For example, the target cancer antigen can be present onthe surface of the cancer cell at a level that is at least 2, 5, 10;100; 1,000; 10,000; 100,000; or 1,000,000-fold higher than acorresponding non-cancer cell. In some cases, the correspondingnon-cancer cell is a cell of the same tissue or origin that is nothyperproliferative or otherwise cancerous. In general, a subject IgGantibody that specifically binds to an antigen (a target antigen) of acancer cell preferentially binds to that particular antigen relative toother available antigens. However, the target antigen need not bespecific to the cancer cell or even enriched in cancer cells relative toother cells (e.g., the target antigen can be expressed by other cells).Thus, in the phrase “an antibody that specifically binds to an antigenof a cancer cell,” the term “specifically” refers to the specificity ofthe antibody and not to the uniqueness of the antigen in that particularcell type.

Modified Fe Region

In some embodiments, the antibodies in the immunoconjugates contain amodified Fc region, wherein the modification modulates the binding ofthe Fc region to one or more Fc receptors.

The terms “Fc receptor” or “FcR” refer to a receptor that binds to theFc region of an antibody. There are three main classes of Fc receptors:FcγR which bind to IgG, FcαR which binds to IgA, and FcεR which binds toIgE. The FcγR family includes several members, such as FcγI (CD64),FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), FcγRIIIB (CD16B).The Fcγ receptors differ in their affinity for IgG and also havedifferent affinities for the IgG subclasses (e.g., IgG1, IgG2, IgG3,IgG4).

In some embodiments, the antibodies in the immunoconjugates (e.g.,antibodies conjugated to a TLR agonist such as a TLR7/8 agonist via alinker) contain one or more modifications (e.g., amino acid insertion,deletion, and/or substitution) in the Fc region that results inmodulated binding (e.g., increased binding or decreased binding) to oneor more Fc receptors (e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB(CD32B), FcγRIIIA (CD16a), and/or FcγRIIIB (CD16b)) as compared to thenative antibody lacking the mutation in the Fc region. In someembodiments, the antibodies in the immunoconjugates contain one or moremodifications (e.g., amino acid insertion, deletion, and/orsubstitution) in the Fc region that reduce the binding of the Fc regionof the antibody to FcγRIIB. In some embodiments, the antibodies in theimmunoconjugates contain one or more modifications (e.g., amino acidinsertion, deletion, and/or substitution) in the Fc region of theantibody that reduce the binding of the antibody to FcγRIIB whilemaintaining the same binding or having increased binding to FcγRI(CD64), FcγRIIA (CD32A), and/or FcRyIIIA (CD16a) as compared to thenative antibody lacking the mutation in the Fc region. In someembodiments, the antibodies in the immunoconjugates contain one of moremodifications in the Fc region that increase the binding of the Fcregion of the antibody to FcγRIIB.

In some cases, the modulated binding is provided by mutations in the Fcregion of the antibody relative to the native Fc region of the antibody.The mutations can be in a CH2 domain, a CH3 domain, or a combinationthereof. A “native Fc region” is synonymous with a “wild-type Fc region”and comprises an amino acid sequence that is identical to the amino acidsequence of an Fc region found in nature or identical to the amino acidsequence of the Fc region found in the native antibody (e.g.,rituximab). Native sequence human Fc regions include a native sequencehuman IgG1 Fc region; native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof. Native sequence Fcincludes the various allotypes of Fcs (see, e.g., Jefferis et al., mAbs,1(4): 332-338 (2009)).

In some embodiments, the mutations in the Fc region that result inmodulated binding to one or more Fc receptors can include one or more ofthe following mutations: SD (S239D), SDIE (S239D/I332E), SE (S267E),SELF (S267E/L328F), SDIE (S239D/I332E), SDIEAL (S239D/I332E/A330L), GA(G236A), ALIE (A330L/I332E), GASDALIE (G236A/S239D/A330L/I332E), V9(G237D/P238D/P271G/A330R), and V11 (G237D/P238D/H268D/P271G/A330R)and/or one or more mutations at the following amino acids: E233, G237,P238, H268, P271, L328 and A330. Additional Fc region modifications formodulating Fc receptor binding are described, e.g., in US PatentApplication Publication 2016/0145350, and U.S. Pat. Nos. 7,416,726 and5,624,821.

In some embodiments, the Fc region of the antibodies of theimmunoconjugates are modified to have an altered glycosylation patternof the Fc region compared to the native non-modified Fc region.

Human immunoglobulin is glycosylated at the Asn297 residue in the Cy2domain of each heavy chain. This N-linked oligosaccharide is composed ofa core heptasaccharide, N-acetylglucosamine4Mannose3 (GlcNAc4Man3).Removal of the heptasaccharide with endoglycosidase or PNGase F is knownto lead to conformational changes in the antibody Fc region, which cansignificantly reduce antibody-binding affinity to activating FcγR andlead to decreased effector function. The core heptasaccharide is oftendecorated with galactose, bisecting GlcNAc, fucose or sialic acid, whichdifferentially impacts Fc binding to activating and inhibitory FcγR.Additionally, it has been demonstrated that u2,6-sialyation enhancesanti-inflammatory activity in vivo while defucosylation leads toimproved FcγRIIIa binding and a 10-fold increase in antibody-dependentcellular cytotoxicity and antibody-dependent phagocytosis. Specificglycosylation patterns can therefore be used to control inflammatoryeffector functions.

In some embodiments, the modification to alter the glycosylation patternis a mutation. For example, a substitution at Asn297. In someembodiments, Asn297 is mutated to glutamine (N297Q). Methods forcontrolling immune response with antibodies that modulate FcγR-regulatedsignaling are described, for example, in U.S. Pat. No. 7,416,726, aswell as US 2007/0014795 and US 2008/0286819.

In some embodiments, the antibodies of the immunoconjugates are modifiedto contain an engineered Fab region with a non-naturally occurringglycosylation pattern. For example, hybridomas can be geneticallyengineered to secrete afucosylated mAb, desialylated mAb ordeglycosylated Fc with specific mutations that enable increased FcRyIIIabinding and effector function. In some embodiments, the antibodies ofthe immunoconjugates are engineered to be afucosylated (e.g.,afucosylated rituximab, available from Invivogen, hcd20-mab13).

In some embodiments, the entire Fc region of an antibody in theimmunoconjugates is exchanged with a different Fc region, so that theFab region of the antibody is conjugated to a non-native Fc region. Forexample, the Fab region of rituximab, which normally comprises an IgG1Fc region, can be conjugated to IgG2, IgG3, IgG4, or IgA, or the Fabregion of nivolumab, which normally comprises an IgG4 Fc region, can beconjugated to IgG1, IgG2, IgG3, IgA1 or IgG2. In some embodiments, theFc modified antibody with a non-native Fc domain also comprises one ormore amino acid modification, such as the S228P mutation within the IgG4Fc, that modulate the stability of the Fc domain described. In someembodiments, the Fc modified antibody with a non-native Fc domain alsocomprises one or more amino acid modifications described herein thatmodulate Fc binding to FcR.

In some embodiments, the modifications that modulate the binding of theFc region to FcR do not alter the binding of the Fab region of theantibody to its antigen when compared to the native non-modifiedantibody. In other embodiments, the modifications that modulate thebinding of the Fc region to FcR also increase the binding of the Fabregion of the antibody to its antigen when compared to the nativenon-modified antibody.

Antibody Targets

In some embodiments, the antibody is capable of binding one or moretargets selected from (e.g., specifically binds to a target selectedfrom) 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A,Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, AMH, AMHR2, ANGPT1,ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, aromatase, ATX, AX1,AZGP1 (zinc-a-glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAG1, BAI1,BCR, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BIyS, BMP1, BMP2, BMP3B(GDFIO), BMP4, BMP6, BMP8, BMPR1A, BMPR1B, BMPR2, BPAG1 (plectin),BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CANT1, CAPRIN-1, CASP1,CASP4, CAV1, CCBP2 (D6/JAB61), CCL1 (1-309), CCLI1 (eotaxin), CCL13(MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC),CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC,exodus-2, CCL22(MDC/STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2/eotaxin-2),CCL25 (TECK), CCL26(eotaxin-3), CCL27 (CTACK/ILC), CCL28, CCL3 (MIP-Ia),CCL4 (MIPIb), CCL5(RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2,CCND1, CCNE1, CCNE2, CCR1 (CKR1/HM145), CCR2 (mcp-IRB/RA), CCR3(CKR3/CMKBR3), CCR4, CCR5(CMKBR5/ChemR13), CCR6(CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBI1), CCR8(CMKBR8/TERI/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR),CD164, CD19, CDIC, CD2, CD20, CD21, CD200, CD-22, CD24, CD27, CD28, CD3,CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD38, CD40, CD40L, CD44,CD45RB, CD47, CD52, CD69, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81,CD83, CD86, CD137, CD152, CD274, CDH1 (Ecadherin), CDH10O, CDH12, CDH13,CDH18, CDH19, CDH20, CDH5, CDH7, CDH8, CDH9, CDK2, CDK3, CDK4, CDK5,CDK6, CDK7, CDK9, CDKN1A (p21Wapl/Cipl), CDKN1B (p27Kipl), CDKN1C,CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CERI, CHGA, CHGB,Chitinase, CHST1O, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7,CKLFSF8, CLDN3, CLDN7 (claudin-7), CLN3, CLU (clusterin), CMKLR1, CMKOR1(RDC1), CNR1, COL18A1, COLIA1, COL4A3, COL6A1, CR2, Cripto, CRP, CSF1(M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTL8, CTNNB1 (b-catenin), CTSB(cathepsin B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL10O(IP-IO), CXCLI1 (1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16,CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9(MIG), CXCR3 (GPR9/CKR-L2), CXCR4, CXCR6 (TYMSTR/STRL33/Bonzo), CYB5,CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DNCL1, DPP4, E2F1, Engel,Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola, ENO2, ENO3,EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10,EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2,EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2,EPHRIN-B3, EPHB4, EPG, ERBB2 (Her-2), EREG, ERK8, Estrogen receptor,Earl, ESR2, F3 (TF), FADD, farnesyltransferase, FasL, FASNf, FCER1A,FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13,FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22,FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8,FGF9, FGFR3, FIGF (VEGFD), FIL1(EPSILON), FBL1 (ZETA), FLJ12584,FLJ25530, FLRT1 (fibronectin), FLT1, FLT-3, FOS, FOSL1(FRA-1), FY(DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GDF5,GFI1, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPR81(FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, HAVCR2, HDAC, HDAC4,HDAC5, HDAC7A, HDAC9, Hedgehog, HGF, HIF1A, HIP1, histamine andhistamine receptors, HLA-A, HLA-DRA, HLA-E, HM74, HMOXI, HSP90,HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-α, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6,BFNA7, IFNB1, IFNgamma, IFNW1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3,IGFBP6, DL-1, ILIO, ILIORA, ILIORB, IL-1, IL1R1 (CD121a), IL1R2(CD121b),IL-IRA, IL-2, IL2RA (CD25), IL2RB(CD122), IL2RG(CD132), IL-4,IL-4R(CD123), IL-5, IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA, (CD126),IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCR1 (IL8RA), CXCR2,(IL8RB/CD128), IL-9, IL9R(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B),IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13,IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C,IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5,IL1F6, ILIF7, IL1F8, DL1F9, ILIHY1, ILIR1, IL1R2, ILIRAP, ILIRAPL1,ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R,IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB,IL2RG, IL3, IL30, IL3RA, IL4, IL4, IL6ST (glycoprotein 130), ILK, INHA,INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (a6integrin), ITGAV, ITGB3, ITGB4 (134 integrin), JAG1, JAK1, JAK3, JTB,JUN, K6HF, KAI1, KDR, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12,KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (Keratin19), KRT2A, KRTHB6(hair-specific type II keratin), LAMA5, LEP (leptin),Lingo-p75, Lingo-Troy, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2,LTBR, MACMARCKS, MAG or OMgp, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine,MIF, MISRII, MJP-2, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3(metallothionectin-UI), mTOR, MTSS1, MUC1 (mucin), MYC, MYD88, NCK2,neurocan, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo),NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCHi, NOX5, NPPB, NROB1,NROB2, NRID1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2,NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3,NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZI, OPRDI, P2RX7, PAP,PART1, PATE, PAWR, PCA3, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB,PECAMI, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PI3Kinase, PIK3CG, PLAU (uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP (CXCL7),PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN,PTGS2 (COX-2), PTN, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGS1,RGS13, RGS3, RNFIIO (ZNF144), Ron, ROBO2, RXR, S100A2, SCGB 1D2(lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1(endothelial Monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3,SERPINB5 (maspin), SERPINEI (PAI-I), SERPINFI, SHIP-1, SHIP-2, SHB1,SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Spr1),ST6GAL1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDGF1, TEK,TGFA, TGFB1, TGFBI1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL,THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissuefactor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11, TNF, TNF-α, TNFAIP2 (B94), TNFAIP3, TNFRSFIiA, TNFRSF1A,TNFRSFiB, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9,TNFSFO1 (TRAIL), TNFSFi 1 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April),TNFSF13B, TNFSF14 (HVEM-L), TNFRSF14 (HVEM), TNFSF15 (VEGI), TNFSF18,TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-likereceptors, TOP2A (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1,TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TRPC6, TSLP,TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4,Wnt-1, XCL1 (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5/CCXCRi), YY1,ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8),CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLECiB(CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), PDGFRa, SLAMF7, GP6(GPVI), LILRAi (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7),LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335, LY94, NKp46),NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1, CD300C,CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS,KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB,SIGLECI (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPB1(CD172B), TREM1 (CD354), TREM2, and KLRF1 (NKp80).

In some embodiments, the antibody binds to an FcRγ-coupled receptor. Insome embodiments, the FcRγ-coupled receptor is selected from the groupconsisting of GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4(CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335,LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, andTARM1.

In some embodiments, the antibody binds to a DAP12-coupled receptor. Insome embodiments, the DAP12-coupled receptor is selected from the groupconsisting of CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D),KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D),NCR2 (CD336, NKp44), PILRB, SIGLECI (CD169, SN), SIGLEC14, SIGLEC15(CD33L3), SIGLEC16, SIRPB1 (CD172B), TREM1 (CD354), and TREM2.

In some embodiments, the antibody binds to a hemITAM-bearing receptor.In some embodiments, the hemITAM-bearing receptor is KLRF1 (NKp80).

In some embodiments, the antibody is capable of binding one or moretargets selected from CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D(MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1,CLECSF5), CLECIB (CLEC-2), CLEC9A (DNGR-1), and CLEC7A (Dectin-1). Insome embodiments, the antibody is capable of binding CLEC6A (Dectin-2)or CLEC5A. In some embodiments, the antibody is capable of bindingCLEC6A (Dectin-2).

In some embodiments, the antibody is capable of binding one or moretargets selected from (e.g., specifically binds to a target selectedfrom): ATP5I (Q06185), OAT (P29758), AIFM1 (Q9ZOX1), AOFA (Q64133), MTDC(P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (088967), LPPRC (Q6PB66),LONM (Q8CGK3), ACON (Q99KI0), ODO1 (Q60597), IDHP (P54071), ALDH2(P47738), ATPB (P56480), AA™ (P05202), TMM93 (Q9CQW0), ERGI3 (Q9CQE7),RTN4 (Q99P72), CL041 (Q8BQR4), ERLN2 (Q8BFZ9), TERA (Q01853), DAD1(P61804), CALX (P35564), CALU (035887), VAPA (Q9WV55), MOGS (Q80UM7),GANAB (Q8BHN3), ERO1A (Q8R180), UGGG1 (Q6P5E4), P4HA1 (Q60715), HYEP(Q9D379), CALR (P14211), AT2A2 (055143), PDIA4 (P08003), PDIA1 (P09103),PDIA3 (P27773), PDIA6 (Q922R8), CLH (Q68FD5), PPIB (P24369), TCPG(P80318), MOT4 (P57787), NICA (P57716), BASI (P18572), VAPA (Q9WV55),ENV2 (P11370), VAT1 (Q62465), 4F2 (P10852), ENOA (P17182), ILK (055222),GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH (Q68FD5), DSG1A(Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75(P38647), ENPL (P08113), CH60 (P63038), and CH10 (Q64433). In thepreceding list, accession numbers are shown in parentheses.

In some embodiments, the antibody binds to an antigen selected fromCDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM, MUC1, MUC16,EGFR, Her2, SLAMF7, and gp75. In some embodiments, the antigen isselected from CD19, CD20, CD47, EpCAM, MUC1, MUC16, EGFR, and Her2. Insome embodiments, the antibody binds to an antigen selected from the Tnantigen and the Thomsen-Friedenreich antigen.

In some embodiments, the antibody or Fc fusion protein is selected from:abagovomab, abatacept (also known as ORENCIA™), abciximab (also known asREOPRO™, c7E3 Fab), adalimumab (also known as HUMIRA™), adecatumumab,alemtuzumab (also known as CAMPATH™, MabCampath or Campath-1H),altumomab, afelimomab, anatumomab mafenatox, anetumumab, anrukizumab,apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab,bapineuzumab, basiliximab (also known as SIMULECT™), bavituximab,bectumomab (also known as LYMPHOSCAN™), belimumab (also known asLYMPHO-STAT-B™), bertilimumab, besilesomab, bevacizumab (also known asAVASTIN™), biciromab brallobarbital, bivatuzumab mertansine, campath,canakinumab (also known as ACZ885), cantuzumab mertansine, capromab(also known as PROSTASCINT™), catumaxomab (also known as REMOVAB™),cedelizumab (also known as CIMZIA™), certolizumab pegol, cetuximab (alsoknown as ERBITUX™), clenoliximab, dacetuzumab, dacliximab, daclizumab(also known as ZENAPAX™), denosumab (also known as AMG 162), detumomab,dorlimomab aritox, dorlixizumab, duntumumab, durimulumab, durmulumab,ecromeximab, eculizumab (also known as SOLIRIS™), edobacomab,edrecolomab (also known as Mabl7-1A, PANOREX™), efalizumab (also knownas RAPTIVA™), efungumab (also known as MYCOGRAB™), elsilimomab,enlimomab pegol, epitumomab cituxetan, efalizumab, epitumomab,epratuzumab, erlizumab, ertumaxomab (also known as REXOMUN™), etanercept(also known as ENBREL™), etaracizumab (also known as etaratuzumab,VITAXIN™, ABEGRIN™), exbivirumab, fanolesomab (also known asNEUTROSPEC™), faralimomab, felvizumab, fontolizumab (also known asHUZAF™), galiximab, gantenerumab, gavilimomab (also known as ABXCBL™),gemtuzumab ozogamicin (also known as MYLOTARG™), golimumab (also knownas CNTO 148), gomiliximab, ibalizumab (also known as TNX-355),ibritumomab tiuxetan (also known as ZEVALIN™), igovomab, imciromab,infliximab (also known as REMICADE™), inolimomab, inotuzumab ozogamicin,ipilimumab (also known as MDX-010, MDX-101), iratumumab, keliximab,labetuzumab, lemalesomab, lebrilizumab, lerdelimumab, lexatumumab (alsoknown as, HGS-ETR2, ETR2-ST01), lexitumumab, libivirumab, lintuzumab,lucatumumab, lumiliximab, mapatumumab (also known as HGSETR1, TRM-1),maslimomab, matuzumab (also known as EMD72000), mepolizumab (also knownas BOSATRIA™), metelimumab, milatuzumab, minretumomab, mitumomab,morolimumab, motavizumab (also known as NUMAX™), muromonab (also knownas OKT3), nacolomab tafenatox, naptumomab estafenatox, natalizumab (alsoknown as TYSABRI™, ANTEGREN™), nebacumab, nerelimomab, nimotuzumab (alsoknown as THERACIM hR3™, THERA-CIM-hR3™, THERALOC™), nofetumomabmerpentan (also known as VERLUMA™), ocrelizumab, odulimomab, ofatumumab,omalizumab (also known as XOLAIR™), oregovomab (also known as OVAREX™),otelixizumab, pagibaximab, palivizumab (also known as SYNAGIS™),panitumumab (also known as ABX-EGF, VECTIBIX™), pascolizumab, pemtumomab(also known as THERAGYN™), pertuzumab (also known as 2C4, OMNITARG™),pexelizumab, pintumomab, priliximab, pritumumab, ranibizumab (also knownas LUCENTIS™), raxibacumab, regavirumab, reslizumab, rituximab (alsoknown as RITUXAN™, MabTHERA™), rovelizumab, ruplizumab, satumomab,sevirumab, sibrotuzumab, siplizumab (also known as MEDI-507),sontuzumab, stamulumab (also known as MYO-029), sulesomab (also known asLEUKOSCAN™), tacatuzumab tetraxetan, tadocizumab, talizumab,taplitumomab paptox, tefibazumab (also known as AUREXIS™), telimomabaritox, teneliximab, teplizumab, ticilimumab, tocilizumab (also known asACTEMRA™), toralizumab, tositumomab, trastuzumab (also known asHERCEPTIN™), tremelimumab (also known as CP-675,206), tucotuzumabcelmoleukin, tuvirumab, urtoxazumab, ustekinumab (also known as CNTO1275), vapaliximab, veltuzumab, vepalimomab, visilizumab (also known asNUVION™), volociximab (also known as M200), votumumab (also known asHUMASPECT™), zalutumumab, zanolimumab (also known as HuMAX-CD4),ziralimumab, zolimomab aritox, daratumumab, elotuxumab, obintunzumab,olaratumab, brentuximab vedotin, afibercept, abatacept, belatacept,afibercept, etanercept, romiplostim, SBT-040 (sequences listed in US2017/0158772. In some embodiments, the antibody is rituximab.

Checkpoint Inhibitors

Any suitable immune checkpoint inhibitor is contemplated for use withthe immunoconjugates disclosed herein. In some embodiments, the immunecheckpoint inhibitor reduces the expression or activity of one or moreimmune checkpoint proteins. In another embodiment, the immune checkpointinhibitor reduces the interaction between one or more immune checkpointproteins and their ligands. Inhibitory nucleic acids that decrease theexpression and/or activity of immune checkpoint molecules can also beused in the methods disclosed herein.

The data herein show that immune checkpoint inhibitor Nivolumab which isnormally an IgG4, can we modified to include an IgG1 Fc, andsubsequently converted into a immunoconjugates of the invention. Thedata indicate that the Nivolumab IgG1 immunoconjugate is still verypotent. Similarly, when the IgG1 NQ Fc on the clinical gradeAtezolizumab was replaced with IgG1, there were improved results. SeeFIGS. 97A-97H.

Most checkpoint antibodies are designed not to have effector function asthey are not trying to kill cells, but rather to block the signalling.Immunoconjugates of the present invention can add back the “effectorfunctionality” needed to activate myeloid immunity. Hence, for mostcheckpoint antibody inhibitors this discovery will be critical.

In some embodiments, the immune checkpoint inhibitor is cytotoxicT-lymphocyte antigen 4 (CTLA4, also known as CD152), T cellimmunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-inducedTNFR-related protein (GITR, also known as TNFRSF18), inducible T cellcostimulatory (ICOS, also known as CD278), CD96, poliovirusreceptor-related 2 (PVRL2, also known as CD112R, programmed cell deathprotein 1 (PD-1, also known as CD279), programmed cell death 1 ligand 1(PD-L1, also known as B7-H3 and CD274), programmed cell death ligand 2(PD-L2, also known as B7-DC and CD273), lymphocyte activation gene-3(LAG-3, also known as CD223), B7-H4, killer immunoglobulin receptor(KIR), Tumor Necrosis Factor Receptor superfamily member 4 (TNFRSF4,also known as OX40 and CD134) and its ligand OX40L (CD252), indoleamine2,3-dioxygenase 1 (IDO-1), indoleamine 2,3-dioxygenase 2 (IDO-2),carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMi), Band T lymphocyte attenuator (BTLA, also known as CD272), T-cell membraneprotein 3 (TIM3), the adenosine A2A receptor (A2Ar), and V-domain Igsuppressor of T cell activation (VISTA protein). In some embodiments,the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, orPD-L1.

In some embodiments, the antibody is selected from: ipilimumab (alsoknown as Yervoy®) pembrolizumab (also known as Keytruda®), nivolumab(also known as Opdivo®), atezolizumab (also known as Tecentrig®),avelumab (also known as Bavencio®), and durvalumab (also known asImfinzi™). In some embodiments, the antibody is selected from:ipilimumab (also known as Yervoy®), pembrolizumab (also known asKeytruda®), nivolumab (also known as Opdivo®), and atezolizumab (alsoknown as Tecentrig®).

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCTLA4. In some embodiments, the immune checkpoint inhibitor is anantibody against CTLA4. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against CTLA4. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstCTLA4. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas CTLA4.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-1. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstPD-1. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas PD-1.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L1. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstPD-L1. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas PD-L1. In some embodiments, the immune checkpoint inhibitor reducesthe interaction between PD-1 and PD-L1.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L2. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L2. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L2. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstPD-L2. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas PD-L2. In some embodiments, the immune checkpoint inhibitor reducesthe interaction between PD-1 and PD-L2.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofLAG-3. In some embodiments, the immune checkpoint inhibitor is anantibody against LAG-3. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against LAG-3. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstLAG-3. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas LAG-3.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofB7-H4. In some embodiments, the immune checkpoint inhibitor is anantibody against B7-H4. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against B7-H4. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstB7-H4. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas B7-H4.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofKIR. In some embodiments, the immune checkpoint inhibitor is an antibodyagainst KIR. In some embodiments, the immune checkpoint inhibitor is amonoclonal antibody against KIR. In some embodiments, the immunecheckpoint inhibitor is a human or humanized antibody against KIR. Insome embodiments, the immune checkpoint inhibitor reduces the expressionor activity of one or more immune checkpoint proteins, such as KIR.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofTNFRSF4. In some embodiments, the immune checkpoint inhibitor is anantibody against TNFRSF4. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against TNFRSF4. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstTNFRSF4. In some embodiments, the immune checkpoint inhibitor reducesthe expression or activity of one or more immune checkpoint proteins,such as TNFRSF4.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofOX40L. In some embodiments, the immune checkpoint inhibitor is anantibody against OX40L. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against OX40L. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstOX40L. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas OX40L. In some embodiments, the immune checkpoint inhibitor reducesthe interaction between TNFRSF4 and OX40L.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofIDO-1. In some embodiments, the immune checkpoint inhibitor is anantibody against IDO-1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against IDO-1. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstIDO-1. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas IDO-1.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofIDO-2. In some embodiments, the immune checkpoint inhibitor is anantibody against IDO-2. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against IDO-2. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstIDO-2. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas IDO-2.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCEACAM1. In some embodiments, the immune checkpoint inhibitor is anantibody against CEACAM1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against CEACAM1. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstCEACAM1. In some embodiments, the immune checkpoint inhibitor reducesthe expression or activity of one or more immune checkpoint proteins,such as CEACAM1.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofBTLA. In some embodiments, the immune checkpoint inhibitor is anantibody against BTLA. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against BTLA. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstBTLA. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas BTLA.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofTIM3. In some embodiments, the immune checkpoint inhibitor is anantibody against TIM3. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against TIM3. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstTIM3. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas TIM3.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofA2Ar. In some embodiments, the immune checkpoint inhibitor is anantibody against A2Ar. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against A2Ar. In some embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstA2Ar. In some embodiments, the immune checkpoint inhibitor reduces theexpression or activity of one or more immune checkpoint proteins, suchas A2Ar.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofVISTA protein. In some embodiments, the immune checkpoint inhibitor isan antibody against VISTA protein. In some embodiments, the immunecheckpoint inhibitor is a monoclonal antibody against VISTA protein. Insome embodiments, the immune checkpoint inhibitor is a human orhumanized antibody against VISTA protein. In some embodiments, theimmune checkpoint inhibitor reduces the expression or activity of one ormore immune checkpoint proteins, such as VISTA protein.

Biosimilars

The immunoconjugates of the present invention are also effective withantibody constructs that are highly similar, or biosimilar, to thecommercially available, or “innovator”, antibody constructs. Forexample, biosimilar antibodies to cetuximab, rituximab, and trastuzumabwere used in several successful immunoconjugates of the presentinvention as seen in FIGS. 71A-71AQ. The biosimilar immunoconjugateselicited myeloid activation as effectively as the commercially availableantibodies. From these studies, it is expected that biosimilarimmunoconjugates will perform similarly to immunoconjugates of theinnovator products.

DAR Ratios

The immunoconjugates of the present invention provide DAR ratios whichare desirable. As seen in FIGS. 84A-87C, the immunoconjugates of thepresent invention provide DAR ratios of 0.7, 1.6, and 2.5.

The immunoconjugates shown with varying DAR ratios were all effective atactivating myeloid cells and eliciting cytokine secretion. The dataindicate that the immunoconjugates with varying DAR ratios were allsuperior at eliciting APC activation as CD40, CD86 and HLA-DR wereexpressed at higher levels in APCs stimulated with immunoconjugates ascompared to those stimulated with the antibody alone. Theimmunoconjugates with varying DARs consistently induced thedownregulation of CD14 and CD16 and increased expression of CD123, ascompared to the antibody alone. From these studies, it is expected allDAR ratios will be effective at eliciting myeloid cell activation.

Isotype Modification

The data herein show (see FIGS. 88C-88H) that when the IgG1 fc region ofantibody, such as rituximab, is exchanged for IgG1 AF, IgG1 NQ, IgG2,IgG3, IgG4, or IgA2, and then formed into an immunoconjugates of thepresent invention, the activity of the immunoconjugate can be modulatedand often, improved, for the desired application.

Around 30% of human IgG is glycosylated within the Fab region, and theantibody in the immunoconjugates of the invention can contain anengineered Fab region with a non-naturally occurring glycosylationpattern. For example, hybridomas can be genetically engineered tosecrete afucosylated mAb, desialylated mAb or deglycosylated Fc withspecific mutations that enable increased FcRyIIIa binding and effectorfunction.

Antibodies for forming immunoconjugates can contain engineered (i.e.,non-naturally occurring) cysteine residues characterized by altered(e.g., enhanced) reactivity toward the reagents used for covalentlybonding the adjuvant moieties to the antibodies. In certain embodiments,an engineered cysteine residue will have a thiol reactivity value in therange of 0.6 to 1.0. In many cases, the engineered antibody will be morereactive than the parent antibody.

In general, the engineered residues are “free” cysteine residues thatare not part of disulfide bridges. The term “thiol reactivity value” isa quantitative characterization of the reactivity of free cysteine aminoacids. As used herein, the term “thiol reactivity value” refers to thepercentage of a free cysteine amino acid in an engineered antibody whichreacts with a thiol-reactive reagent, and converted to a maximum valueof 1. For example, a cysteine residue in an engineered antibody whichreacts in 100% yield with a thiol-reactive reagent, such as a maleimide,to form a modified antibody has a thiol reactivity value of 1.0. Anothercysteine residue engineered into the same or different parent antibodywhich reacts in 80% yield with a thiol-reactive reagent has a thiolreactivity value of 0.8. Determination of the thiol reactivity value ofa particular cysteine residue can be conducted by ELISA assay, massspectroscopy, liquid chromatography, autoradiography, or otherquantitative analytical tests.

Engineered cysteine residues can be located in the antibody heavy chainsor the antibody light chains. In certain embodiments, engineeredcysteine residues are located in the Fc region of the heavy chains. Forexample, amino acid residues at positions L-15, L-43, L-110, L-144,L-168 in the light chains of an antibody or H-40, H-88, H-119, H-121,H-122, H-175, and H-179 in the heavy chains of an antibody can bereplaced with cysteine residues. Ranges within about 5 amino acidresidues on each side of these positions can also be replaced withcysteine residues, i.e., L-10 to L-20; L-38 to L-48; L-105 to L-115;L-139 to L-149; L-163 to L-173; H-35 to H-45; H-83 to H-93; H-114 toH-127; and H-170 to H-184, as well as the ranges in the Fc regionselected from H-268 to H-291; H-319 to H-344; H-370 to H-380; and H-395to H-405, to provide useful cysteine engineered antibodies for formingimmunoconjugates. Other engineered antibodies are described, forexample, in U.S. Pat. Nos. 7,855,275; 8,309,300; and 9,000,130, whichare hereby incorporated by reference.

In addition to antibodies, alternative protein scaffolds may be used aspart of the immunoconjugates. The term “alternative protein scaffold”refers to a non-immunoglobulin derived protein or peptide. Such proteinsand peptides are generally amenable to engineering and can be designedto confer monospecificity against a given antigen, bispecificity, ormultispecificity. Engineering of an alternative protein scaffold can beconducted using several approaches. A loop grafting approach can be usedwhere sequences of known specificity are grafted onto a variable loop ofa scaffold. Sequence randomization and mutagenesis can be used todevelop a library of mutants, which can be screened using variousdisplay platforms (e.g., phage display) to identify a novel binder.Site-specific mutagenesis can also be used as part of a similarapproach. Alternative protein scaffolds exist in a variety of sizes,ranging from small peptides with minimal secondary structure to largeproteins of similar size to a full-sized antibody. Examples of scaffoldsinclude, but are not limited to, cystine knotted miniproteins (alsoknown as knottins), cyclic cystine knotted miniproteins (also known ascyclotides), avimers, affibodies, the tenth type III domain of humanfibronectin, DARPins (designed ankyrin repeats), and anticalins (alsoknown as lipocalins). Naturally occurring ligands with known specificitycan also be engineered to confer novel specificity against a giventarget. Examples of naturally occurring ligands that may be engineeredinclude the EGF ligand and VEGF ligand. Engineered proteins can eitherbe produced as monomeric proteins or as multimers, depending on thedesired binding strategy and specificities. Protein engineeringstrategies can be used to fuse alternative protein scaffolds to Fcdomains.

Preparation of Antibody Adjuvant Conjugates

Reactions for forming the immunoconjugates of the invention areconducted under conditions sufficient to covalently bond the adjuvantmoiety to the antibody. In general, the reactions are conducted bycontacting an antibody with an adjuvant-linker compound such that anamino acid sidechain in the antibody reacts with the adjuvant linkercompound. In some embodiments, the adjuvant-linker compound and theantibody are used in approximately equimolar amounts when forming theimmunoconjugates. In some embodiments, an excess of the adjuvant-linkercompound is used when forming the immunoconjugates. For example, areaction mixture for forming an immunoconjugate can contain from about1.1 to about 50 molar equivalents of the adjuvant-linker compound withrespect to the antibody.

The reactions can be conducted at any suitable temperature. In general,the reactions are conducted at a temperature of from about 4° C. toabout 40° C. The reactions can be conducted, for example, at about 25°C. or about 37° C. The reactions can be conducted at any suitable pH. Ingeneral, the reactions are conducted at a pH of from about 4.5 to about10. The reactions can be conducted, for example, at a pH of from about 5to about 9. In some embodiments, the reaction is conducted at nearneutral pH (i.e., around pH 7). In some embodiments, the reaction isconducted at a pH ranging from 7.2 to 7.5. The reactions can beconducted for any suitable length of time. In general, the reactionmixtures are incubated under suitable conditions for anywhere betweenabout 1 minute and several hours. The reactions can be conducted, forexample, for about 1 minute, or about 5 minutes, or about 10 minutes, orabout 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours,or about 8 hours, or about 12 hours, or about 24 hours, or about 48hours, or about 72 hours. Other reaction conditions may be employed inthe methods of the invention, depending on the identity of the antibodyin the conjugate and the reagent used for installing the adjuvantmoiety.

Reaction mixtures for forming the antibody adjuvant conjugates cancontain additional reagents of the sort typically used in bioconjugationreactions. For example, in certain embodiments, the reaction mixturescan contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),3-morpholinopropane-1-sulfonic acid (MOPS), potassium phosphate, sodiumphosphate, phosphate-buffered saline, sodium citrate, sodium acetate,and sodium borate), cosolvents (e.g., dimethylsulfoxide,dimethylformamide, ethanol, methanol, tetrahydrofuran, acetone, andacetic acid), salts (e.g., NaCl, KCl, CaCl₂, and salts of Mn²⁺ andMg²⁺), detergents/surfactants (e.g., a non-ionic surfactant such asN,N-bis[3-(D-gluconamido)propyl]cholamide, polyoxyethylene (20) cetylether, dimethyldecylphosphine oxide, branched octylphenoxypoly(ethyleneoxy)ethanol, a polyoxyethylene-polyoxypropylene blockcopolymer, t-octylphenoxypolyethoxyethanol, polyoxyethylene (20)sorbitan monooleate, and the like; an anionic surfactant such as sodiumcholate, N-lauroylsarcosine, sodium dodecyl sulfate, and the like; acationic surfactant such as hexdecyltrimethyl ammonium bromide,trimethyl(tetradecyl) ammonium bromide, and the like; or a zwitterionicsurfactant such as an amidosulfobetaine,3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate, and thelike), chelators (e.g., ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),2-({2-[bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid(EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid(BAPTA)), and reducing agents (e.g., dithiothreitol (DTT),β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)).Buffers, cosolvents, salts, detergents/surfactants, chelators, andreducing agents can be used at any suitable concentration, which can bereadily determined by one of skill in the art. In general, buffers,cosolvents, salts, detergents/surfactants, chelators, and reducingagents are included in reaction mixtures at concentrations ranging fromabout 1 μM to about 1 M. For example, a buffer, a cosolvent, a salt, adetergent/surfactant, a chelator, or a reducing agent can be included ina reaction mixture at a concentration of about 1 μM, or about 10 μM, orabout 100 μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M.

Formulation and Administration of Immunoconjugates

In a related aspect, the invention provides a composition comprising aplurality of immunoconjugates as described above. In some embodiments,the average number of adjuvant moieties per immunoconjugate ranges fromabout 1 to about 10. The average number of adjuvant moieties perimmunoconjugate can range, for example, from about 1 to about 10, orfrom about 1 to about 6, or from about 1 to about 4. The average numberof adjuvant moieties per immunoconjugate can be about 0.8, 1, 1.2, 1.4,1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4.0, or 4.2. Insome embodiments, the average number of adjuvant moieties perimmunoconjugate is about 4. In some embodiments, the average number ofadjuvant moieties per immunoconjugate is about 2. In some cases, theantibody is covalently bonded to a single adjuvant moiety. In somecases, the antibody is covalently bonded to 2 or more adjuvant moieties(e.g., 3 or more, 4 or more, or 5 or more adjuvant moieties). In somecases, the antibody is covalently bonded to 1-10 adjuvant moieties(e.g., 1-8, 1-5, 1-3, 2-10, 2-8, 2-5, 2-3, or 3-8 adjuvant moieties). Insome cases, the antibody is covalently bonded to 2-10 adjuvant moieties(e.g., 2-8, 2-5, 2-3, or 3-10, or 3-8 adjuvant moieties). In some casesin which the antibody is covalently bonded to more than one adjuvantmoiety, the attached adjuvant moieties can be the same or different. Forexample, in some cases two or more of the adjuvant moieties can be thesame (e.g., two different molecules of the same adjuvant moiety can eachbe attached to the antibody at a different site on the antibody). Insome cases, the antibody is covalently bonded to 2 or more differentadjuvant moieties (e.g., 3 or more, 4 or more, or 5 or more differentadjuvant moieties). For example, when generating an immunoconjugate ofthe invention, one or more antibodies can be contacted with a mixturethat includes two or more (e.g., 3 or more, 4 or more, or 5 or more)different adjuvant-linker compounds such that amino acid sidechains inthe one or more antibodies reacts with the adjuvant-linker compounds,thus resulting in one or more immunoconjugates that are each covalentlybonded to two or more different adjuvant moieties.

Site-specific antibody conjugation allows for precise placement of theadjuvant on the antibody and a homogenous DAR as compared to theheterogeneous conjugation product resulting from attachment to lysineresidues in the antibody. Site-specific immunoconjugates may begenerated through various modifications of the antibody. Methods forsite-specific conjugation include the following methods but are notlimited to those methods described herein. One method for site-specificconjugation involves the incorporation of a sequence that is thenrecognized by an enzyme, resulting in chemical modification. Forexample, the enzyme FGE recognizes the sequence Cys-X-Pro-X-Arg.Co-expression of the modified antibody along with FGE in mammalianculture generates an antibody containing an aldehyde-tag at theengineered site(s). Other enzymes may be used that recognize naturallyoccurring sequences or residues for conversion to chemically reactivegroups allowing for site-specific conjugation. Bacterialtransglutaminases (BTGs) can catalyze the formation of bonds betweenglutamine residues and primary amines; the bacterial enzyme sortase Acan catalyze transpeptidation reactions through a recognition motif.Non-natural amino acids may also be incorporated into the antibodysequence that may then be reacted to generate site-specific conjugates.Naturally occurring residues, such as the amino acid selenocysteine, maybe incorporated into the antibody and subsequently reacted with theappropriate reactive groups including but not limited to maleimides andiodoacetamides for site-specific conjugation. Another method is theincorporation of engineered cysteine residues that are added into theheavy or light chain of the antibody construct. Vectors encoding for theheavy and/or light chains are modified to incorporate the codon sequencefor a cysteine residue (vector sequence in FIGS. 138A-138B and vectormap in FIGS. 138C-138D). Conjugation is performed by first reducing theantibody and then re-oxidizing to regenerate the native disulfide bondsof the antibody, resulting in the uncapping of a reactive thiol(s). Oncereacted with adjuvant-linker, the resulting product contains ahomogenous population of immunoconjugate with a DAR defined by thenumber of cysteine residues engineered into the antibody (structureshown in FIG. 138E). For example, the incorporation of a mutation in thelight chain at position 205 from a valine to cysteine (V205C mutation)results in a product with the adjuvant conjugated at the defined sites(V205C; FIGS. 138F-138G).

In some embodiments, the composition further comprises one or morepharmaceutically acceptable excipients. For example, theimmunoconjugates of the invention can be formulated for parenteraladministration, such as intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Alternatively,the immunoconjugates can be injected intra-tumorally. Formulations forinjection will commonly comprise a solution of the immunoconjugatedissolved in a pharmaceutically acceptable carrier. Among the acceptablevehicles and solvents that can be employed are water and Ringer'ssolution, an isotonic sodium chloride. In addition, sterile fixed oilscan conventionally be employed as a solvent or suspending medium. Forthis purpose, any bland fixed oil can be employed including syntheticmonoglycerides or diglycerides. In addition, fatty acids such as oleicacid can likewise be used in the preparation of injectables. Thesesolutions are sterile and generally free of undesirable matter. Theseformulations can be sterilized by conventional, well known sterilizationtechniques. The formulations can contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents,e.g., sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of theimmunoconjugate in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight, andthe like, in accordance with the particular mode of administrationselected and the patient's needs. In certain embodiments, theconcentration of an immunoconjugate in a solution formulation forinjection will range from about 0.1% (w/w) to about 10% (w/w).

In another aspect, the invention provides a method for treating cancer.The method includes comprising administering a therapeutically effectiveamount of an immunoconjugate (e.g., as a composition as described above)to a subject in need thereof. For example, the methods can includeadministering the immunoconjugate to provide a dose of from about 100ng/kg to about 50 mg/kg to the subject. The immunoconjugate dose canrange from about 5 mg/kg to about 50 mg/kg, from about 10 ag/kg to about5 mg/kg, or from about 100 ag/kg to about 1 mg/kg. The immunoconjugatedose can be about 100, 200, 300, 400, or 500 ag/kg. The immunoconjugatedose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. Theimmunoconjugate dose can also lie outside of these ranges, depending onthe particular conjugate as well as the type and severity of the cancerbeing treated. Frequency of administration can range from a single doseto multiple doses per week, or more frequently. In some embodiments, theimmunoconjugate is administered from about once per month to about fivetimes per week. In some embodiments, the immunoconjugate is administeredonce per week.

Some embodiments of the invention provide methods for treating cancer asdescribed above, wherein the cancer is a head and neck cancer. Head andneck cancer (as well as head and neck squamous cell carcinoma) refers toa variety of cancers characterized by squamous cell carcinomas of theoral cavity, pharynx and larynx, salivary glands, paranasal sinuses, andnasal cavity, as well as the lymph nodes of the upper part of the neck.Head and neck cancers account for approximately 3 to 5 percent of allcancers in the United States. These cancers are more common in men andin people over age 50. Tobacco (including smokeless tobacco) and alcoholuse are the most important risk factors for head and neck cancers,particularly those of the oral cavity, oropharynx, hypopharynx andlarynx. Eighty-five percent of head and neck cancers are linked totobacco use. In the methods of the invention, the immunoconjugates canbe used to target a number of malignant cells. For example, theimmunoconjugates can be used to target squamous epithelial cells of thelip, oral cavity, pharynx, larynx, nasal cavity, or paranasal sinuses.The immunoconjugates can be used to target mucoepidermoid carcinomacells, adenoid cystic carcinoma cells, adenocarcinoma cells, small-cellundifferentiated cancer cells, esthesioneuroblastoma cells, Hodgkinlymphoma cells, and Non-Hodgkin lymphoma cells. In some embodiments,methods for treating head and neck cancer include administering animmunoconjugate containing an antibody that is capable of binding EGFR(e.g., cetuximab, panitumumab, matuzumab, and zalutumumab), PD-1 (e.g.,pembrolizumab), and/or MUC1.

Some embodiments of the invention provide methods for treating cancer asdescribed above, wherein the cancer is breast cancer. Breast cancer canoriginate from different areas in the breast, and a number of differenttypes of breast cancer have been characterized. For example, theimmunoconjugates of the invention can be used for treating ductalcarcinoma in situ; invasive ductal carcinoma (e.g., tubular carcinoma;medullary carcinoma; mucinous carcinoma; papillary carcinoma; orcribriform carcinoma of the breast); lobular carcinoma in situ; invasivelobular carcinoma; inflammatory breast cancer; and other forms of breastcancer. In some embodiments, methods for treating breast cancer includeadministering an immunoconjugate containing an antibody that is capableof binding HER2 (e.g., trastuzumab, margetuximab), glycoprotein NMB(e.g., glembatumumab), and/or MUC1.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedherein may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-98 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

1. An immunoconjugate comprising

(a) an antibody construct comprising (i) an antigen binding domain and(ii) an Fc domain,

(b) an adjuvant moiety, and

(c) a linker,

wherein each adjuvant moiety is covalently bonded to the antibodyconstruct via the linker.

2. The immunoconjugate of aspect 1, wherein the antibody constructfurther comprises a targeting binding domain.

3. The immunoconjugate of aspect 1, wherein the antibody construct is anantibody.

4. The immunoconjugate of any one of aspects 1-3, wherein the antigenbinding domain binds to an antigen of a cancer cell.

5. The immunoconjugate of any one of aspects 1-4, wherein the antigenbinding domain binds to an antigen selected from the group consisting ofCDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM, MUC1, MUC16,EGFR, VEGF, HER2, SLAMF7, PDGFRa, and gp75.

6. The immunoconjugate of any one of aspects 1-5, wherein the antigenbinding domain binds to an antigen selected from the group consisting ofCD19, CD20, CD40, CD47, EpCAM, MUC1, MUC16, PDGFRa, EGFR, and HER2.

7. The immunoconjugate of any one of aspects 1-6, wherein the antigenbinding domain binds to an antigen selected from the group consisting ofTn antigen and the Thomsen-Friedenreich antigen.

8. The immunoconjugate of any one of aspects 3-7, wherein the antibodyis a polyclonal antibody.

9. The immunoconjugate of any one of aspects 3-7, wherein the antibodyis a monoclonal antibody.

10. The immunoconjugate of aspect 8 or 9, wherein the antibody ishumanized.

11. The immunoconjugate of aspect 8 or 9, wherein the antibody ismurine.

12. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis selected from the group consisting of olaratumab, obinutuzumab,trastuzumab, cetuximab, rituximab, pertuzumab, bevacizumab, daratumumab,etanercept, and elotuzumab.

13. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis olaratumab.

14. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis obinutuzumab.

15. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis trastuzumab.

16. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis cetuximab.

17. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis rituximab.

18. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis pertuzumab.

19. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis bevacizumab.

20. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis daratumumab.

21. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis elotuzumab.

22. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis etanercept.

23. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds to an antigen of an immune checkpoint inhibitor.

24. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds to an antigen selected from the group consisting of CTLA4, PD-1,PD-L1, PD-L2, LAG-3, B7-H4, KIR, TNFRSF4, OX40L, IDO-1, IDO-2, CEACAMi,BTLA, TIM3, A2Ar, and VISTA.

25. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds to an antigen selected from the group consisting of CTLA4, PD-1,and PD-L1.

26. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds a PD-1 antigen.

27. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds a PD-L1 antigen.

28. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds a CTLA4 antigen.

29. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis selected from the group consisting of pembrolizumab, nivolumab,atezolizumab, and ipilimumab.

30. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis pembrolizumab.

31. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis nivolumab.

32. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis atezolizumab.

33. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis ipilimumab.

34. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds to an antigen selected from the group consisting of CLEC4C(BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle),CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLECiB (CLEC-2), CLEC9A(DNGR-1), and CLEC7A (Dectin-1).

35. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds to CLEC5A.

36. The immunoconjugate of any one of aspects 3-11, wherein the antibodybinds to CLEC6A (Dectin-2).

37. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis an IgA1.

38. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis an IgA2 antibody.

39. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis an IgG antibody.

40. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis an IgG1 antibody.

41. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis an IgG2 antibody.

42. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis an IgG3 antibody.

43. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis an IgG4 antibody.

44. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis a biosimilar of an antibody selected from the group consisting ofpembrolizumab, nivolumab, atezolizumab, ipilimumab obinutuzumab,trastuzumab, cetuximab, rituximab, pertuzumab, bevacizumab, daratumumab,etanercept olaratumab, and elotuzumab.

45. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis a biosimilar of cetuximab.

46. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis a biosimilar of rituximab.

47. The immunoconjugate of any one of aspects 3-11, wherein the antibodyis a biosimilar of trastuzumab.

48. The immunoconjugate of any one of aspects 3-11, wherein the antibodycomprises a modified Fc region.

49. The immunoconjugate of aspect 48, wherein the modified Fc regioncontains at least one amino acid insertion, deletion, or substitution.

50. The immunoconjugate of aspect 48, wherein the modified Fc regionresults in modulated binding of an Fc receptor selected from the groupconsisting of FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA(CD16a), and FcγRIIIB (CD16b), as compared to the native antibodylacking the modified Fc region.

51. The immunoconjugate of aspect 48, wherein the modified Fc regionincreases the binding of the Fc region to an Fc receptor FcγRIIIA(CD16a).

52. The immunoconjugate of aspect 48, wherein the modified Fc regionincreases the binding of the Fc region to an Fc receptor FcγRIIIB (CD16b).

53. The immunoconjugate of any one of aspects 1-52, wherein theimmunoconjugate has a structure according to Formula I:

or a pharmaceutically acceptable salt thereof, wherein Ab is anantibody; A is an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; Z is a linking moiety;Adj is an adjuvant moiety; and subscript r is an integer from 1 to 10.

54. The immunoconjugate of aspect 53, wherein the immunoconjugate has astructure according to Formula Ia:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Ab is an antibody;    -   A is an unmodified amino acid sidechain in the antibody or a        modified amino acid sidechain in the antibody;    -   Z is a linking moiety;    -   R¹ is selected from H and C₁₋₄ alkyl; or    -   Z, R¹, and the nitrogen atom to which they are attached form a        linking moiety comprising a 5- to 8-membered heterocycle;    -   each Y is independently CHR², wherein R² is selected from H, OH,        and NH₂,    -   R³ is selected from C₁₋₆ alkyl and 2- to 6-membered heteroalkyl,        each of which is optionally substituted with one or more members        selected from the group consisting of halo, hydroxy, amino, oxo        (═O), alkylamino, amido, acyl, nitro, cyano, and alkoxy;    -   X is selected from O and CH₂;    -   subscript n is an integer from 1 to 12; and    -   subscript r is an integer from 1 to 10.

55. The immunoconjugate of aspect 54, wherein the immunoconjugate has astructure according to Formula Ib:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Ab is an antibody;    -   A is an unmodified amino acid sidechain in the antibody or a        modified amino acid sidechain in the antibody;    -   Z is a linking moiety;    -   R¹ is selected from H and C₁₋₄ alkyl; or    -   Z, R¹, and the nitrogen atom to which they are attached form a        linking moiety comprising a 5- to 8-membered heterocycle;    -   each Y is independently CHR², wherein R² is selected from H, OH,        and NH₂;    -   X is selected from O and CH₂;    -   subscript n is an integer from 1 to 12; and    -   W is selected from the group consisting of O and CH₂.

56. The immunoconjugate of aspect 55, wherein the immunoconjugate has astructure according to Formula Ic:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Ab is an antibody;    -   subscript r is an integer from 1 to 10;    -   A is an unmodified amino acid sidechain in the antibody or a        modified amino acid sidechain in the antibody;    -   Z is a linking moiety; and    -   R¹ is selected from H and C₁₋₄ alkyl; or    -   Z, R¹, and the nitrogen atom to which they are attached form a        linking moiety comprising a 5- to 8-membered heterocycle; and    -   R² is selected from H, OH, and NH₂.

57. The immunoconjugate of aspect 56, the immunoconjugate has astructure according to Formula Id:

or a pharmaceutically acceptable salt thereof, wherein Ab is anantibody; A is an unmodified amino acid sidechain in the antibody or amodified amino acid sidechain in the antibody; R² is selected from H,OH, and NH₂; and subscript r is an integer from 1 to 10.

58. The immunoconjugate of any one of aspects 53-56, wherein Z isselected from:

wherein subscript x is an integer from 1 to 12; subscript y is aninteger from 1 to 30; the dashed line (“

”) represents the point of attachment to the adjuvant moiety; and thewavy line (“

”) represents the point of attachment to an amino acid sidechain in theantibody.

59. The immunoconjugate of any one of aspects 1-52, wherein theimmunoconjugate has a structure according to Formula II:

or a pharmaceutically acceptable salt thereof, wherein Ab is anantibody; wherein A is an unmodified amino acid sidechain in theantibody or a modified amino acid sidechain in the antibody; wherein Adjis an adjuvant moiety; wherein subscript r is an integer 1 to 10; andwherein:

-   -   Z¹ is selected from —C(O)—, —C(O)NH—, —CH₂—;    -   Z² and Z⁴ are independently selected from a bond, C₁₋₃₀        alkylene, and 3- to 30-membered heteroalkylene, wherein:        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by —C(O)—, —NR^(a)C(O)—, or            —C(O)NR^(a)-,        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            carbocycle,        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            heterocycle having one to four heteroatoms selected from O,            S, and N, and        -   each R^(a) is independently selected from H and C₁₋₆ alkyl;    -   Z³ is selected from a bond, a divalent peptide moiety, and a        divalent polymer moiety; and    -   Z⁵ is bonded to the sidechain of an amino acid sidechain in the        antibody.

60. The immunoconjugate of aspect 59, wherein the immunoconjugate has astructure according to Formula IIa:

-   -   or a pharmaceutically acceptable salt thereof, wherein    -   Z¹ is selected from —C(O)—, —C(O)NH—, —CH₂—;    -   Z² and Z⁴ are independently selected from a bond, C₁₋₃₀        alkylene, and 3- to 30-membered heteroalkylene, wherein:        -   one or more groupings of adjacent atoms in the C₁₋₃₀ alkyl            and 3- to 30-membered heteroalkylene are optionally and            independently replaced by —C(O)—, —NR^(a)C(O)—, or            —C(O)NR^(a)—;        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            carbocycle,        -   one or more groupings of adjacent atoms in the C₁₋₃₀            alkylene and 3- to 30-membered heteroalkylene are optionally            and independently replaced by a 4- to 8-membered, divalent            heterocycle having one to four heteroatoms selected from O,            S, and N, and        -   each R^(a) is independently selected from H and C₁₋₆ alkyl;    -   Z³ is selected from a bond, a divalent peptide moiety, and a        divalent polymer moiety; and    -   Z⁵ is selected from an amine-bonded moiety and a thiol-bonded        moiety.

61. The immunoconjugate of any one of aspects 1-52, wherein theimmunoconjugate has a structure according to Formula III:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain, Adj is an adjuvant, G is CH₂, C═O,or a bond, L is a linker, and subscript r is an integer from 1 to 10.

62. The immunoconjugate of aspect 61, wherein L is selected from:

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; a is an integerfrom 1 to 40; each A is independently selected from any amino acid;subscript c is an integer from 1 to 20; the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to

63. The immunoconjugate of aspect 61, wherein the immunoconjugate has astructure according to Formula IIIa-Formula IIIg:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; G is CH₂, C═O,or a bond; R is optionally present and is a linear or branched, cyclicor straight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbon units; subscript a is aninteger from 1 to 40; each A is independently selected from any aminoacid; subscript c is an integer from 1 to 20; and subscript r is aninteger from 1 to 10.

64. The immunoconjugate of any one of aspects 61-63, wherein theimmunoconjugate has a structure according to Formula IVa-Formula IVk:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain; Adj is an adjuvant; and subscript ris an integer from 1 to 10.

65. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is a pattern recognition receptor (PRR)agonist.

66. The immunoconjugate of aspect 65, wherein the adjuvant moiety is aToll-like receptor (TLR) agonist.

67. The immunoconjugate of aspect 65, wherein the adjuvant moiety is aToll-like receptor (TLR) agonist selected from the group consisting of aTLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR7 agonist, a TLR8agonist, a TLR7/TLR8 agonist, and a TLR9 agonist.

68. The immunoconjugate of aspect 65, wherein the adjuvant moiety is aTLR7 agonist, a TLR8 agonist, or a TLR7/TLR8 agonist.

69. The immunoconjugate of aspect 65, wherein the adjuvant moiety isselected from the group consisting of gardiquimod(1-(4-amino-2-ethylaminomethylimidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol),imiquimod (R837), loxoribine, IRM1(1-(2-amino-2-methylpropyl)-2-(ethoxymethyl)-1H-imidazo-[4,5-c]quinolin-4-amine),IRM2(2-methyl-1-[2-(3-pyridin-3-ylpropoxy)ethyl]-1H-imidazo[4,5-c]quinolin-4-amine),IRM3 (N-(2-[2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy]ethyl)-N-methylcyclohexanecarboxamide),CL097 (2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine), CL307,CL264, resiquimod, 3M-052/MEDI9197, SD-101(N-[(4S)-2,5-dioxo-4-imidazolidinyl]-urea), motolimod(2-amino-N,N-dipropyl-8-[4-(pyrrolidine-1-carbonyl)phenyl]-3H-1-benzazepine-4-carboxamide),CL075 (2-propylthiazolo[4,5-c]quinolin-4-amine), and TL8-506(3H-1-benzazepine-4-carboxylic acid, 2-amino-8-(3-cyanophenyl)-, ethylester), N-a-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine,palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl) (Pam3Cys), triacyl lipidA (OM-174), Lipoteichoic acid (LTA), peptidoglycan, CL419(S-(2,3-bis(palmitoyloxy)-(2RS)propyl)-(R)-cysteinyl spermine), Pam₂CSK₄(S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysinex 3 CF3COOH), CL572 (S-(2-myristoyloxy ethyl)-(R)-cysteinyl4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl) aniline),CL413(S-(2,3-bis(palmitoyloxy)-(2RS)propyl)-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysyl4-((6-amino-2-(butylamino)-8-hydroxy-9H-purin-9-yl)methyl)aniline), andCL401 (S-(2,3-bis(palmitoyloxy)-(2RS)propyl)-(R)-cysteinyl4-((6-amino-2(butyl amino)-8-hydroxy-9H-purin-9-yl)methyl) aniline).

70. The immunoconjugate of aspect 65, wherein the adjuvant is animidazoquinoline compound.

71. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein each J independently is hydrogen, OR⁴, or R⁴; each R⁴independently is hydrogen, or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 carbon units; Q is optionally present and is analkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,arylalkyl, or heteroarylalkyl group comprising from 1 to 8 carbon units;and the dashed line (“

”) represents the point of attachment of the adjuvant.

72. The immunoconjugate of aspect 71, wherein the adjuvant moiety is offormula:

wherein each R⁴ independently is selected from the group consisting ofhydrogen, or alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl group comprising from 1 to 8carbon units and the dashed line (“

”) represents the point of attachment of the adjuvant.

73. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein J is hydrogen, OR⁴, or R⁴; each R⁴ independently is hydrogen, oralkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,arylalkyl, and heteroarylalkyl group comprising from 1 to 8 carbonunits; Q is selected from the group consisting of alkyl, or heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, andheteroarylalkyl group comprising from 1 to 8 carbon units; and thedashed line (“

”) represents the point of attachment of the adjuvant.

74. The immunoconjugate of aspect 72, wherein the adjuvant moiety is offormula:

wherein each R⁴ independently is selected from the group consisting ofhydrogen, or alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, arylalkyl, and heteroarylalkyl group comprising from 1 to 8carbon units and the dashed line (“

”) represents the point of attachment of the adjuvant.

75. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein each R⁴ independently is hydrogen, or alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, orheteroarylalkyl group comprising from 1 to 8 carbon units; Q is alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl,or heteroarylalkyl group comprising from 1 to 8 carbon units; and thedashed line (“

”) represents the point of attachment of the adjuvant.

76. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein each J independently is hydrogen, OR⁴, or R⁴; each R⁴independently is hydrogen, or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 carbon units; each U independently is CH or Nwherein at least one U is N; each subscript t independently is aninteger from 1 to 3; Q is optionally present and is an alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl,or heteroarylalkyl group comprising from 1 to 8 carbon units; and thedashed line (“

”) represents the point of attachment of the adjuvant.

77. The immunoconjugate of aspect 74, wherein the adjuvant moiety is offormula:

wherein R⁴ is selected from the group consisting of hydrogen, or alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl,and heteroarylalkyl group comprising from 1 to 8 carbon units Q is analkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,arylalkyl, or heteroarylalkyl group comprising from 1 to 8 carbon units;and the dashed line (“

”) represents the point of attachment of the adjuvant.

78. The immunoconjugate of any one of aspects 1-53, 59, and 61-63,wherein the adjuvant moiety is of formula:

wherein J is hydrogen, OR⁴, or R⁴; each R⁴ independently is hydrogen, oran alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,arylalkyl, or heteroarylalkyl group comprising from 1 to 8 carbon units;R⁵ is hydrogen, or an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, arylalkyl, or heteroarylalkyl group comprising from 1to 10 carbon units; Q is an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 carbon units; and the dashed line (“

”) represents the point of attachment of the adjuvant.

79. The immunoconjugate of aspect 76, wherein the adjuvant moiety is offormula:

wherein J is hydrogen, OR⁴, or R⁴; each R⁴ independently is selectedfrom the group consisting of hydrogen, or alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, andheteroarylalkyl group comprising from 1 to 8 carbon units; U is CH or N;V is CH₂, O, or NH; each subscript t independently is an integer from 1to 3; and the dashed line (“

”) represents the point of attachment of the adjuvant.

80. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein R¹ is selected from H and C₁₋₄ alkyl; R³ is selected from C₁₋₆alkyl and 2- to 6-membered heteroalkyl, each of which is optionallysubstituted with one or more members selected from the group consistingof halo, hydroxy, amino, oxo (═O), alkylamino, amido, acyl, nitro,cyano, and alkoxy; X is selected from O and CH₂; each Y is independentlyCHR², wherein R² is selected from H, OH, and NH₂, subscript n is aninteger from 1 to 12; and the dashed line (“

”) represents the point of attachment of the adjuvant.

81. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein W is selected from the group consisting of O and CH₂; R¹ isselected from H and C₁₋₄ alkyl; each Y is independently CHR², wherein R²is selected from H, OH, and NH₂; subscript n is an integer from 1 to 12;and the dashed line (“

”) represents the point of attachment of the adjuvant.

82. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein W is selected from the group consisting of O and CH₂; R¹ isselected from H and C₁₋₄ alkyl; each Y is independently CHR², wherein R²is selected from H, OH, and NH₂; subscript n is an integer from 1 to 12;and the dashed line (“

”) represents the point of attachment of the adjuvant.

83. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein W is selected from the group consisting of O and CH₂; X isselected from O and CH₂; each Y is independently CHR², wherein R² isselected from H, OH, and NH₂; subscript n is an integer from 1 to 12;and the dashed line (“

”) represents the point of attachment of the adjuvant.

84. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein R¹ is selected from H and C₁₋₄ alkyl; R² is selected from H, OH,and NH₂; and the dashed line (“

”) represents the point of attachment of the adjuvant.

85. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is of formula:

wherein R¹ is selected from H and C₁₋₄ alkyl; R² is selected from H, OH,and NH₂; and the dashed line (“

”) represents the point of attachment of the adjuvant.

86. The immunoconjugate of any one of aspects 1-53, 59, and 61-64,wherein the adjuvant moiety is:

wherein the dashed line (“

”) represents the point of attachment of the adjuvant.

87. An immunoconjugate selected from:

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibodywith at least one lysine side chain and subscript r is an integer from 1to 10.

88. The immunoconjugate of aspect 87, wherein r is from 1 to 4.

89. The immunoconjugate of any one of aspects 1-88, wherein the linkeris from about 2.5 Å to about 45 Å.

90. The immunoconjugate of any one of aspects 1-88, wherein the linkeris from about 2.5 Å to about 20 Å.

91. A composition comprising a plurality of immunoconjugates accordingto any one of aspects 1-90.

92. The composition of aspect 91, wherein the average number of adjuvantmoieties per immunoconjugate ranges from about 1 to about 9.

93. The composition of aspect 91, wherein the average number of adjuvantmoieties per immunoconjugate ranges from about 0.5 to about 4.

94. The composition of any one of aspects 91-93, further comprising oneor more pharmaceutically acceptable excipients.

95. A method for treating cancer comprising administering atherapeutically effective amount of an immunoconjugate according to anyone of aspects 1-90 or a composition according to any one of aspects91-94 to a subject in need thereof.

96. The method of aspect 95, wherein the cancer is breast cancer.

97. The method of aspect 95, wherein the cancer is a head and neckcancer.

98. The method of aspect 95, wherein the cancer is a lymphoma.

EXAMPLES Example 1. Imidazoquinolines for Antibody Conjugation

Imidazoquinoline compounds with a free amine group (Compound 1) or amaleimide group (Compound 2) were synthesized according to Scheme 1,allowing for the rapid assessment of linker technology andantibody-adjuvant immunoconjugate efficacy.

To determine if adjuvant functionalization impacted the capacity ofCompound 2 or Compound 1 to elicit immune activation, human antigenpresenting cells were stimulated with 10-fold serial dilutions of R848,Compound 2, Compound 1 or a control TLR agonist, CL307, for 18 hoursprior to analysis via flow cytometry. The data indicated that Compound 2and Compound 1 performed similarly to R848 across each concentrationassayed (FIG. 4; Compound 1 data not shown).

Next, the capacity of each functionalized TLR agonist to activate humanTLR7 or TLR8 was directly assayed. HEK293 cells were co-transfected withhuman TLR7 or TLR8 or murine TLR7 and an inducible secreted embryonicalkaline phosphatase reporter gene under the control of the IFN-βminimal promoter fused to NF-κB and AP-1 binding sites. Cells weresubsequently incubated with 2-fold serial dilutions of each theindicated adjuvants for 12 hours at 37 C in the presence of an alkalinephosphatase substrate. Activity was measured by spectrophotometry (OD650 nm). The data indicate that Compound 1 activated both human TLR7 andTLR8 whereas Compound 2 was specific for TLR7 activity (FIG. 2).Similarly, both Compound 2 and Compound 1 activated murine TLR7 (FIG.2).

Example 2. Preparation of Antibody Adjuvant Conjugates

Compound 1 was modified with a non-cleavable crosslinker (SMCC,ThermoFisher Scientific) and a cleavable crosslinker (SPDP, ThermoFisherScientific) in preparation for conjugation to rituximab according to thegeneral scheme outlined in Scheme 2A and Scheme 2B.

Adjuvants with a free amine (R848, Compound 1, etc.) were conjugated toSMCC, SPDP or other NHS containing linkers by reacting the compounds ata 1:1 molar ratio in PBS or other suitable buffers at pH 7-7.5. Allreactions were protected from light and incubated for 30 minutes at roomtemperature. Where possible, adjuvant-crosslinker conjugates werepurified via reverse phase high-pressure liquid chromatography (HPLC).Adjuvant-crosslinker conjugates were utilized immediately followingconjugation, as described below.

Adjuvant-linker combinations were desalted and buffer exchanged intodeionized water with Zeba Spin Desalting Columns (ThermoFisherScientific). Samples were subsequently analyzed on a Shimadzu LC/MS-2020Single Quadrupole Liquid Chromatograph Mass Spectrometer. A method witha gradient ranging from 0 to 100% acetonitrile suitable for detection ofsmall molecules within 100-1000 m/z was utilized for compound detection.

The reaction efficiency was assessed via LC-MS and indicated that themajority of free SMCC had reacted with Compound 1 to form Compound1-SMCC, which had the expected molecular weight of 531 (FIG. 3, lowerright panel). Similar reaction efficiencies were observed with Compound1-SPDP (data not shown).

Following the successful conjugation of Compound 1 to the crosslinkers,antibodies were modified with the SATA crosslinker to convert the freeamines on the antibody to protected sulfhydryl groups. Followingconjugation of SATA, sulfhydryl groups were deacetylated withhydroxylamine and exposed thiols were reacted with the maleimidecomponent of the adjuvant-SMCC compound as shown in Scheme 3 of FIG.139.

Antibody was resuspended in phosphate buffered saline (PBS) at 1-5 mg/mLand the SATA crosslinker (ThermoFisher Scientific) was resuspended at 70mM in anhydrous DMSO immediately before usage. Antibody was reacted witha 10-fold molar excess of SATA at room temperature for 30 minutes. TheSATA-modified antibody was purified from excess reagent and byproductswith 3 washes in PBS with equilibrated Amicon Ultra Centrifugal FilterUnits with Ultracel-100 membranes according to the manufacturer'sinstructions (EMD Millipore). The number of SATA crosslinkers perantibody was determined by matrix-assisted laser desorption/ionizationmass spectrometry (MALDI-TOF).

SATA-modified antibody was deacetylated following a 2-hour incubation atroom temperature in PBS at pH between 7.2-7.5 with 0.05 M hydroxylamineand 2.5 mM EDTA. The deacetylated SATA-modified antibody wassubsequently purified from excess reagent and byproducts with 3 washesin PBS containing 5 mM EDTA with equilibrated Amicon Ultra CentrifugalFilter Units with Ultracel-100 membranes according to the manufacturer'sinstructions (EMD Millipore). Purified deacetylated SATA-modifiedantibody was subsequently reacted with a 5 to 40-fold molar excess ofadjuvant-crosslinker for 30 minutes to one hour at room temperature. Theexact molar excess was 10-fold higher than the average number of SATAmolecules per antibody as determined by MALDI-TOF. Followingconjugation, the antibody adjuvant immunoconjugate was purified fromexcess reagent and byproducts with 3 washes in PBS with equilibratedAmicon Ultra Centrifugal Filter Units with Ultracel-100 membranesaccording to the manufacturer's instructions (EMD Millipore).

The average drug to antibody ratio was determined via MALDI-TOF. Sampleswere desalted and buffer exchanged using Zeba Spin Desalting Columns(ThermoFisher Scientific) into deionized water. Matrix (sinapinic acid)was first spotted onto the MALDI sample target plate and allowed to dry.Next, the sample was mixed at a 1:1 ratio with and without a bovineserum albumin (BSA) standard (0.25-1 μM BSA) and spotted onto the platewith the matrix samples. Once both the matrix and sample layer dried,samples were analyzed on a AB Sciex TOF/TOF 5800 (Stanford University,Canary Center). A high mass detector (CovalX) with negative ionizationallowed for enhanced sensitivity and resolution at protein sizes in therange of a fully intact IgG antibody (˜150,000 kDa).

Following successful conjugation of the antibody-adjuvantimmunoconjugate (Ab-SATA-SMCC-Adjuvant shown in Scheme 3 of FIG. 139),the average drug to antibody ratio was determined via MALDI-TOF massspectrometry (Table 1). The mass difference between the SATA-modifiedand unmodified antibody was utilized to determine how many linkers werepresent per antibody. The mass difference between the SATA-modifiedantibody and the immunoconjugates were utilized to determine the averagedrug to antibody ratio (DAR).

TABLE 1 MALDI-TOF MS-based determination of Drug-to-Antibody Ratio.Molecular Mass Weight Difference Ab Sample (Da) (Da) ModificationAntibody 145,772 — — Antibody-SATA 146,210 438 3.77 Linkers/AbAntibody-SATA-SPDP- 146,944 1172 2.07 Drugs/Ab Compound 1Antibody-SATA-SMCC- 147,309 1537 2.07 Drugs/Ab Compound 1

Example 3. Assessment of Antibody Adjuvant Conjugate Activity In Vitro

Isolation of Human Antigen Presenting Cells.

Human antigen presenting cells (APCs) were negatively selected fromhuman peripheral blood mononuclear cells obtained from healthy blooddonors (Stanford Blood Center) by density gradient centrifugation usinga RosetteSep Human Monocyte Enrichment Cocktail (Stem Cell Technologies)containing monoclonal antibodies against CD2, CD3, CD8, CD19, CD56,CD66b and CD235a. Immature APCs were subsequently purified to >97%purity via negative selection using an EasySep Human Monocyte EnrichmentKit without CD16 depletion containing monoclonal antibodies against CD2,CD3, CD19, CD20, CD56, CD66b, CD123 and CD235a.

Preparation of Tumor Cells.

Tumor cells were resuspended in PBS with 0.1% fetal bovine serum (FBS)at 1 to 10×10⁶ cells/mL. Cells were subsequently incubated with 2 μMCFSE to yield a final concentration of 1 μM. The reaction was endedafter 2 minutes via the addition of 10 mL complete medium with 10% FBSand washed once with complete medium. Cells were either fixed in 2%paraformaldehyde and washed three times with PBS or left unfixed priorto freezing the cells in 10% DMSO, 20% FBS and 70% medium.

APC-Tumor Co-Cultures.

2×10⁵ APCs were incubated with or without 6.5×10⁵ autologous orallogeneic CFSE-labeled tumor cells in 96-well plates (Corning)containing IMDM medium (Gibco) supplemented with 10% fetal bovine serum,100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, sodiumpyruvate, non-essential amino acids, 50 μM 2-ME and, where indicated,various concentrations of anti-tumor antibody and the indicatedadjuvants. Cells and cell-free supernatants were analyzed after 18 hoursvia flow cytometry.

Results.

To determine the capacity of immunoconjugates to elicit immuneactivation, human APCs (˜95% monocytes) obtained from fresh blood wereincubated with CFSE-labeled human B cell lymphoma cells (Toledo, ATCC)at a 3:1 ratio and 2-fold serial dilutions of Compound 1, Rituximab(Ab), Rituximab+Compound 1 (Mixture) or Rituximab-SATA-SMCC-Compound 1(conjugated). In these experiments, the immunoconjugate had an averageof 2.1 Compound 1 molecules per antibody and the Compound 1 doses wereadjusted accordingly to ensure that equimolar amounts of Compound 1 werecompared across all conditions. After 18 hours, cells were analyzed forthe expression of activation markers via flow cytometry. The dataindicate that immunoconjugates were far superior at eliciting APCactivation as CD40, CD86 and HLA-DR were expressed at several foldhigher levels in APCs stimulated with the immunoconjugate as compared tothose stimulated with Ab alone, Compound 1 alone or the mixture (FIG.4).

Given the high level of activation markers observed followingimmunoconjugate activation, the expression of PD-L1, an inhibitorymarker that is highly correlated with the extent of APC activation, wasinvestigated. Surprisingly, immunoconjugates were much less potent ateliciting the upregulation of PD-L1 expression as compared to theadjuvant alone or the mixture (FIG. 5). Notably, PD-L1 expression wasnegligible at 0.1 μM immunoconjugate, which corresponds to the maximallybioactive concentration (FIG. 4, FIG. 5). These data suggest that theimmunoconjugate may activate unforeseen signaling pathways in humanAPCs.

In support of this hypothesis, cells stimulated with the immunoconjugateunexpectedly developed dendrites and underwent morphologic changesconsistent with monocytes differentiating into DCs. This findingprompted the analysis of DC associated surface molecules. Consistentwith their morphology, APCs stimulated with the immunoconjugate, but notthe mixture, downregulated CD14, CD16 and CD163 expression in a dosedependent manner (FIG. 6). The downregulation of these molecules, whichare expressed by monocytes and macrophages, but greatly diminished onmonocyte-derived DCs, indicates that human monocytes exposed toimmunoconjugate rapidly differentiated into DCs. Consistent with thesedata, APCs stimulated with the immunoconjugate upregulated theexpression of CD123, a marker of human inflammatory monocyte-derived DCs(FIG. 6).

While the expression of T cell stimulatory molecules such as CD40, CD86and HLA-DR is necessary for effective T cell activation, APCs alsoinfluence the nature of the ensuing immune response through thesecretion of cytokines. Therefore, the capacity of immunoconjugates toelicit cytokine secretion in human APCs following stimulation wasinvestigated as described above. The data indicate that theimmunoconjugate-differentiated cells secreted several fold higheramounts of IL-13 and TNFα whereas secretion of the anti-inflammatorycytokine IL-10 trended lower (FIG. 7).

Immunoconjugates constructed with cleavable linkers have also beenprepared and found to elicit APC activation and DC differentiation invitro (FIG. 8). Human APCs that were ˜95% monocytes were stimulated with2-fold serial dilutions of Rituximab-SATA-SPDP-Compound 1 (conjugated,cleavable), Rituximab alone (Ab), Compound 1 alone or Rituximab+Compound1 (Mixture) in the presence of CFSE-labeled tumor cells.immunoconjugate—Cleavable had a DAR of 1.4 as confirmed by MALDI-TOF.After 18 hours, CD19⁻ human APCs were analyzed via flow cytometry; n=3.

Example 4. Assessment of Antibody Adjuvant Conjugate Efficacy In Vivo

For tumor studies, 2×10⁵ B 16F10 melanoma cells were injectedsubcutaneously (s.c.) above the right flank in C57BL/6 mice. After tendays, or when the tumors reached 25 mm², mice were administeredintravenous injections of 400 μg the immunconjugate(anti-GP75-SATA-SMCC-Compound 1) (DAR=1.74) or treated intratumorallywith 400 μg of the immunoconjugate (anti-GP75-SATA-SMCC-Compound 1) or amixture of 1.5 μg of Compound 1 and 400 μg anti-GP75 (TA99). Subsequenttreatments were administered on days 2 and 4 after the initialtreatment. Tumor development was measured 2-3 times per week withcalipers.

Mice treated with the immunoconjuage, but not the mixture, reduced theirtumors (FIG. 9A). Next, equivalent doses of αGP75-immunoconjugate wereadministered intratumorally or intravenously in mice with establishedtumors. Surprisingly, IV administration resulted in tumor regressioneven though it is estimated that less than 10% of the immunoconjugatereached the tumor (FIG. 9B).

The studies described herein demonstrate that immunoconjugates arequantitatively and qualitatively more effective at eliciting immuneactivation and anti-tumor immunity than equimolar quantities ofnon-covalently attached antibody-adjuvant mixtures. These findings areunlikely to result from simple serum half-life extension of the adjuvantfollowing antibody conjugation, because profound phenotypic alterationsand novel biology were observed during short in vitro incubationperiods. These studies indicate that freshly isolated peripheral bloodmonocytes from healthy human donors undergo DC differentiation followingovernight stimulation with immunoconjugates whereas gold standard DCdifferentiation protocols with GM-CSF and IL-4 require six days.Furthermore, immunoconjugate activated human APCs expressed several foldhigher amounts of co-stimulatory molecules and inflammatory cytokinesthan achievable with equivalent doses of non-covalently attachedantibody-adjuvant mixtures. Yet, immunoconjugates elicit much lowerlevels of negative co-stimulatory molecules such as PD-L1 and comparableamounts of IL-10, suggesting that immunoconjugates activate unforeseensignaling pathways. Without wishing to be bound by any particulartheory, it is believed that stimulation with immunoconjugates closelyresembles physiologic antibody-mediated immunity whereby APCs recognizeopsonized pathogens (antibody bound to pathogens) with high affinity.

Example 5. Preparation and Assessment of Additional Antibody AdjuvantConjugate Activity In Vitro

Preparation of Additional Antibody Adjuvant Conjugates.

Additional antibody-adjuvant conjugates were prepared using the methodsdescribed in Examples 1 and 2. The antibodies pembrolizumab (PD-1),nivolumab (PD-1), atezolizumab (PD-L1), and ipilimumab (CTLA4) were usedto create the antibody-adjuvant conjugates with SATA-SMCC linkers (seeScheme 3 of FIG. 139).

Following successful conjugation of the immunoconjugate, the averagedrug to antibody ratio was determined via LC-MS. The immunoconjugate isfirst deglycosylated using PNGase F to remove glycans from the antibody,and then the immunoconjuage is buffer exchanged into deionized water.Antibody adjuvant conjugates were run on a C4 column eluted withacetonitrile/water on a Waters Xevo G2-XS QTof/Tof. Raw massspectrometry data was deconvoluted to determine the Drug to Antibody(DAR) ratios. The LC-MS data indicated successful conjugation anddesirable DAR ratios.

Isolation of Human Antigen Presenting Cells.

Human antigen presenting cells (APCs) were negatively selected fromhuman peripheral blood mononuclear cells obtained from healthy blooddonors (Stanford Blood Center) by density gradient centrifugation usinga RosetteSep Human Monocyte Enrichment Cocktail (Stem Cell Technologies)containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123,and HLA-DR. Immature APCs were subsequently purified to >97% purity vianegative selection using an EasySep Human Monocyte Enrichment Kitwithout CD 16 depletion containing monoclonal antibodies against CD14,CD16, CD40, CD86, CD123, and HLA-DR.

Preparation of Tumor Cells.

Tumor cells were prepared in accordance with Example 3 above.

APC-Tumor Co-Cultures.

2×10⁵ APCs were incubated with or without 6.5×10⁵ autologous orallogeneic CFSE-labeled tumor cells in 96-well plates (Corning)containing IMDM medium (Gibco) supplemented with 10% fetal bovine serum,100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, sodiumpyruvate, non-essential amino acids, 50 μM 2-ME and, where indicated,various concentrations of antibody. Cells and cell-free supernatantswere analyzed after 18 hours via flow cytometry.

Results.

Human APCs (˜95% monocytes) obtained from fresh blood were incubatedwith CFSE-labeled human B cell lymphoma cells (Toledo, ATCC) at a 3:1ratio and 2-fold serial dilutions of the antibody alone or theAntibody-SATA-SMCC-Compound 1 (conjugated). After 18 hours, cells wereanalyzed for the expression of activation markers via flow cytometry.The data indicate that immunoconjugates were superior at eliciting APCactivation as CD40, CD86, and HLA-DR tended to be expressed at higherlevels in APCs stimulated with the immunoconjugate as compared to thosestimulated with the antibody alone (see FIGS. 10D and 10E forIpilimumab, 11D and 11E for pembrolizumab, 12D and 12E for nivolumab,and 13D and 13E for atezolizumab). Consistent with the results observedin Example 3, the immunoconjugates downregulated CD14 (see FIG. 10C foripilimumab, 11C for pembrolizumab, 12C for nivolumab, and 13C foratezolizumab). The results for these immunoconjugates were as expectedfor CD16 and CD123 (data not shown) based on the results in Example 3.

The capacity of these immunoconjugates to elicit cytokine secretion inhuman APCs following stimulation was investigated as described inExample 3 above. The data indicate that immunoconjugate-differentiatedcells secreted higher amounts of IL-1 and TNFα (see FIGS. 14A and 14Bfor atezolizumab, 15A and 15B for nivolumab, 16A and 16B forpembrolizumab, and 20 for ipilimumab).

Example 6. Preparation and Assessment of Anti-Dectin-2 AdjuvantConjugate Activity In Vitro

Preparation of Additional Antibody Adjuvant Conjugates.

An additional antibody-adjuvant conjugate was prepared using the methodsdescribed in Examples 1 and 2. An anti-Dectin-2 antibody (CLEC6A) andisotype rat IgG2a was used to create the antibody-adjuvant conjugatewith SATA-SMCC linkers (see Scheme 3 of FIG. 139).

The capacity of this immunoconjugate to elicit cytokine secretion inmurine monocyte derived APCs following stimulation was investigated asdescribed in Example 3 above. Specifically, cytokine production is shownin FIG. 21 for GM-CSF-pretreated monocytes that were stimulated for 18hours with the immunoconjugates or equivalent amounts of theunconjugated components. The data indicate thatimmunoconjugate-differentiated cells secreted higher amounts of TNFα,IL-6, and IL-12p70 (see FIG. 21) than equivalent amounts of thecomponents (adjuvant alone, and antibody alone, and control antibodyconjugate).

Dectin-2 and CLEC5A are C-type lectin receptors that associate with andsignal through the adaptor proteins FcRγ (FCER1G) and DAP12 (TYROBP),respectively, following receptor crosslinking. These adaptor proteinscontain immunoreceptor tyrosine-based activation motifs (ITAMs) thatmediate downstream signaling through a Syk-dependent pathway, leading toimmune cell activation (i.e. cytokine production, costimulatory moleculeexpression, antigen presentation, etc.). As shown (FIG. 21 and FIG. 23),immunoconjugates directed against these receptors exhibit synergisticimmunostimulatory effects through simultaneous engagement of theITAM-coupled receptor (through the antigen binding domain) and othersignaling pathways (through the adjuvant moiety, e.g. TLR7/8).Immunoconjugates targeting other receptors that associate with FcRγand/or DAP12, or that contain similar signaling domains (e.g. hemITAM),may be prepared in a similar fashion and are expected to exhibit similareffects.

Example 7. Synthesis of a TLR7/8 Adjuvant

The following steps were taken to prepare a TLR7/TLR8 adjuvant (Scheme1, Compound 1) suitable for conjugation to an antibody to form animmunoconjugate of the present invention. Masses of products wereconfirmed on a UPLC system (Waters Acquity) equipped with a Xevo XS QToFspectrometer detector. Samples dissolved in acetonitrile:water wereinjected onto a BEH200 C18 column (2.1 mm diameter x 50 mm length)eluted with a 10-90% gradient of acetonitrile:water over 5 minutes.

Chilled (00 C) nitric acid (70%, 160 mL) was slowly added to thequinoline-2,4-diol I (100 g 621 mmol) in glacial acetic acid (600 mL)stirring in ice bath. Removed mixture from ice bath then warmed to roomtemperature. Stirred at room temperature for 30 min. Heated at 80° C.for 1.5 hours then cooled the mixture to 0° C. Slowly added 1 L of waterto the mixture to precipitate yellow solid. Stirred vigorously for 15minutes then filtered. Resuspended the solid in water (1 L) and stirredvigorously for 15 minutes then filtered. Repeated with the additionalstep of slowly adding solid NaHCO₃ to bring pH to >6 then suctionfiltered overnight. Resuspended solid in ethyl ether (750 mL) andstirred vigorously to create fine suspension. Filtered and repeated.Suction filtered overnight to dry. Yield 112 g II (88%) yellow solid.

At room temperature, slowly added disopropylethylamine (63 mL, 47 g,0.36 mol, 2.5 eq.) to POCl₃ (300 mL). Heated mixture to 80° C. underblanket of Ar. Slowly added in 2 g portions nitro-diol II (30 g, 145mmol, 1 eq.) over 30 minutes maintaining temperature below 95° C. Afteraddition is completed, raise temperature to 110° C. and heat for 1 hour.Cooled reaction to 0° C. then slowly pour in parts over ice whilevigorously stirring. Added cold water to final volume of 1.2 L thenstirred vigorously. Decanted the aqueous mother liquor and added 1 Lwater to the dark solid, scraping the sticky solid from walls of flaskto create suspension. Repeated as necessary to obtain solid that can befiltered. Resuspended the solid in 1 L water then slowly added solidNaHCO3until pH >6. Filtered the solid then dissolved in EtOAc (500 mL).Filtered EtOAc solution through Celite to remove insoluble blackimpurity. Washed filtrate with saturated NaHCO3, water, brine thenseparated and dried organic layer with Na₂SO₄, filtered and concentratedin vacuo. The brown solid that is formed was trituated with 3:1hexanes/diethyl ether (500 mL), filtered. The tan solid III (22 g, 30mmol, 62%) was used as is in the next reaction.

To a solution of nitro-dichloro compound III (22 g, 62 mmol, 1 eq.) andsolid K₂CO₃ (17 g, 124 mmol, 2 eq.) in DMF (250 mL) at 0° C. was slowlyadded a solution of N-Boc-1,4-diaminobutane (12.8 g, 1.1 eq.) in DMF (60mL) over 30 minutes. After addition was complete the reaction was warmedto room temperature and stirred for an additional 30 minutes. Water (800mL) was added and the mixture was stirred vigorously. The supernatantwas poured off and the wet solid was dissolved in ethyl acetate (500mL). The solution was washed with water, brine, separated, dried(Na₂SO₄), filtered and concentrated in vacuo. The brown solid wastrituated with 1:1 hexanes/diethyl ether (400 mL) and filtered to obtaina yellow solid IV (17 g, 43 mmol, 69%) that was used as in in the nextreaction.

To a solution of nitro-amino compound IV (17 g, 43 mmol, 1 eq.) inmethanol (400 mL) and water (60 mL) at 0° C. was added NiCl₂.6H₂O (0.51g, 2.2 mmol, 0.05 eq). Sodium borohydride (pellets, 3.2 g, 86 mmol, 2eq.) was added and reaction was stirred for 1 h at 0° C. then warmed toroom temperature and allowed to stir for another 15 minutes. Glacialacetic acid was added in parts to neutralize any unreacted NaBH₄ until apH of ˜5 was obtained. The solution was filtered through a bed of Celiteto remove black insoluble material. The solvent was removed in vacuo.The dark brown solid was trituated with ether then filtered to obtain atan solid V (13.3 g, 37 mmol, 85%) that was used as is in the nextreaction.

To a solution of diamino compound V (13.3 g, 37 mmol, 1 eq.) in DMF (250mL) containing disopropylethylamine (7.17 g, 9.7 mL, 56 mmol, 1.5 eq.)stirring at room temperature was added neat valeroyl chloride (5.5 mL,5.5 g, 42 mmol, 1.2 eq). The mixture was stirred for 30 minutes then icewas and then water was added to a final volume of 1 L. The mixture wasstirred vigorously until a clear supernatant was formed. The supernatantwas poured off and the crude solid was dissolved in ethyl acetate (400mL) and filtered through a bed of Celite. The filtrate was washed withwater (400 mL), brine (400 mL), separated then dried (Na2SO4), filteredand concentrated. The solid was trituated with ether, filtered andsuction dried. The brown solid obtained VI (13.9 g, 31 mmol, 84%) wasused in the next reaction as is.

In a 500 mL round bottomed flask equipped with a Dean-Stark apparatus amixture of amide VI (13.9 g, 31 mmol, 1 eq.) and 2-chlorobenzoic (2.4 g,15.5 mmol. 0.5 eq.) was refluxed in 150 mL toluene (bathtemperature=170° C.) for 4 hours. The Dean-Stark apparatus and condenserwas removed and until 80-90% of the toluene was evaporated.2,4-dimethoxybenzylamine (25 g, 150 mmol, 5 eq.) was added and thereaction was continually heated at 120° C. for 1.25 hours. The reactionwas cooled and the crude mixture was diluted with 1:1 MeOH/water (1 L)and vigorously stirred. The supernatant was decanted (removing most ofthe excess 2,4-dimethoxybenzylamine) and the crude product waspartitioned between between water and ethyl acetate. Acetic acid wasadded until the aqueous layer gave a pH of 5-6. The organic layer waswashed with water, brine, dried (Na₂SO₄), filtered and concentrated. Thethick brown syrup was dissolved in diethyl ether and filtered to removea gray solid (not product). The ether was removed to give a brown syrup(14.4 g, 26 mmol, 73%) and was used as is in the next reaction.

To material VII (14.4 g, 26 mmol, 1 eq.) was added water (60 mL) andslowly with swirling conc. HCl (60 mL). The mixture was vigorouslystirred at room temperature for 30 minutes then heated to reflux for 1hour. The reaction was cooled in an ice bath and solid NaOH pellets (28g, 700 mmol) were added in parts over 30 minutes until a basic pH wasachieved. The solution was warmed to room temperature and stirredvigorously. Solid NaCl was added until a saturated solution wasachieved. This aqueous layer was extracted 3 times with 10%isopropanol/dichloromethane (400 mL). The combined organic layers weredried (Na₂SO₄), filtered and concentrated to yield a brown solid VIIIwas obtained (6.8 g, 22 mmol, 79%).

Example 8. Immunoconjugate Synthesis

This example provides guidance on synthesis of an immunoconjugate usingthe TFP ester method. Compound VIII (311 mg, 1 mmol) was dissolved in 10mL of dimethylformamide (DMF) and then 2 molar equivalents ofdiisopropylethylamine (DIPEA) was added. An SMCC linker (1.5 mmol) wasdissolved in 10 mL of dichloromethane and added in one portion to VIII.The reaction was stirred overnight at 20° C. and concentrated to drynessvia rotary evaporation. The crude product IX was purified on a silicagel using a Buchi flash chromatography system loaded with a 12 gdisposable cartridge and eluted with a gradient of 0-10% methanol over15 minutes. Pure fractions were combined and evaporated to dryness toprovidel60 mg of a pale yellow solid IX.

Compound IX (0.1 mmol, 53 mg) was dissolved in 10 mL of dichloromethaneand then 2 equivalents of thioglycolic acid were added at one time. Themixture was concentrated to dryness under vacuum and the residue waswashed three times with 5 mL of diethyl ether.

Compound X (6.2 mg, 0.01 mmol) was dissolved in 2 mL of THF and then 5mg of tetrafluorophenol was added. Then 5 mg of dicyclohexylcarbodiimide(DCC) was added. The mixture was stirred overnight at room temperatureand then concentrated to dryness under vacuum. The crude product XI waspurified via flash chromatography on silica gel (4 gram prepackedcolumn) and eluted with 0-10% MeOH in dichloromethane. Pure fractionswere combined and evaporated to provide 3.6 mg of pure XI (confirmed byLC/MS). The TFP ester XI was then used in the antibody conjugation stepdepicted in Scheme 14 of FIG. 140.

An IgG1 antibody (specifically, the anti-CD20 antibody rituxumab) wasbuffer exchanged into PBS at a pH of 7.2 and diluted to 10 mg/mL (66μM). The TFP activated adjuvant, XI, was added to DMSO and 6 molarequivalents (relative to IgG) was added to 1 mL of the antibody solution(10 mg) in one portion. The mixture was inverted several times to mixand incubated overnight at 20° C. The resulting immunoconjugate(“BB-01”) was purified via buffer exchange into PBS (pH 7.2) using aPD10 column (SephadexG25®) size exclusion chromatography column. Purefractions were pooled and the concentration determined by measuring theabsorbance at 280 nm on a nano-drop spectrophotometer. The yield was 8milligram or approximately 80% based on recovered protein. Theimmunoconjugate product was sterile filtered through a 0.2 am syringefilter and stored at 4° C. until needed.

Characterization of the resulting immunoconjugate's drug to antibodyratio (“DAR”) was performed via liquid chromatography-mass spectrometry(“LC/MS”) analysis on a UPLC system (Waters Aquity) equipped with a XevoXS QToF mass spectrometer detector. Analysis was performed via injectionof 5 ag of the immunoconjugate onto a BEH200 C4 column (2.1 mmdiameter×50 mm length) eluted with a 10-90% gradient ofacetonitrile:water over 4 minutes.

The analysis indicated that the immunoconjugates synthesized via the TFPmethod demonstrated higher DAR than the immunoconjugate synthesizedusing the SATA method. In addition the TFP method yieldedimmunoconjugates with reduced amounts of unconjugated antibody (onlyabout 5%) compared to the SATA synthesis method (about 20%) (compareFIGS. 1A and 1B).

Size exclusion chromatography (“SEC”) analysis of BB-01 was performed todetermine the monomeric purity. Analysis was performed on a BEH200 SECcolumn eluted with PBS (pH 7.2) and 0.2 mL/min. The immunoconjugateBB-01 synthesized using the TFP active ester method contained less than2% of high molecular weight aggregate (FIG. 2B) compared to greater than8% aggregate observed when the SATA method was used (FIG. 2A).

Example 9. Synthesis of Immunoconjugate BB-14 with a Pentafluorophenyl(“PFP”) Ester

This example provides guidance on synthesis of an immunoconjugate usingthe PFP ester method. Ester modification of the adjuvant and conjugationof the modified adjuvant to the antibody is shown in Scheme 15 of FIG.14I. Cyclohexane trans-1,4-dicarboxylate (1 g) was dissolved in 10 mL ofdimethylformamide (“DMF”) and1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate) (“HATU”) (1 mmol) was added followed by 1 mLof N-ethyl-N-(propan-2-yl)propan-2-amine (“DIPEA”). Compound 1 (311 mg)was added and the mixture stirred overnight at 20° C. The reactionmixture was diluted with 50 mL of dichloromethane (“DCM”) and washedwith 20 mL of iN HCl. The DCM layer was evaporated to dryness and theproduct purified on silica gel eluted with 0-10% MeOH in DCM containing1% acetic acid. Pure fractions were concentrated to provide 220 mg ofpurified acid II. Compound II (100 mg) was dissolved in THF and 100 mgof HATU was added followed by 200 aL of DIPEA. Two equivalents ofamino-PEG2-tertbutyl-carboxylate was added and stirred for one hour at20° C. The mixture was concentrated to dryness and 10 milliliters of 4NHCl in dioxane was added. The mixture was concentrated to dryness andthe crude product III was purified by prep HPLC to provide 40 mg ofcompound III.

Compound III was converted to PFP ester IV as described below. CompoundIII (35 mg) was added to 50 mg of PFP in 5 mL THF and 5 mL DMF was addedfollowed by 20 mg of DCC. DMAP (2-3 mg) was added and the solution wasstirred overnight at 20° C. The reaction was concentrated and purifiedby flash chromatography (eluted with 0-10% MeOH) to provide 17 mg of PFPester IV after lyophillization from 1:2 acetonitrile water.

PFP ester IV (6 molar eq. relative to IgG) was added to 20 mg of an IgGantibody (specifically, the anti-CD20 antibody rituximab) (10 mg/mL inPBS) and incubated at 37° C. overnight. The resulting immunoconjugateBB-14 was buffer exchanged into PBS (pH 7.2) to remove excess smallmolecular weight reagent and the concentration determined on thenanodrop. The yield was 15 mg of immunoconjugate (75% yield). Theproduct was stored at 4° C. A DAR of 2.2 was determined via LC/MSanalysis. Besides the desirable DAR and high yield, the product also hadfew impurities as determined by SEC analysis (see FIGS. 3 and 4).

Example 10. Synthesis of Immunoconjugate BB-15 with a NHS Ester

Ester modification of the adjuvant and conjugation of the modifiedadjuvant to the antibody is shown in Scheme 16 of FIG. 142. Compound VII(150 mg) was dissolved in 20 mL of tetrahydrofluran (“THF”) and 10 mL ofaqueous, saturated sodium bicarbonate was added. Then, 50 mg of succinicanhydride was added in one portion and the mixture was stirred for onehour at room temperature. Twenty milliliters of iN HCl was added slowlyand the mixture was extracted with 2×50 mL of dichloromethane. Thecombined organic extracts were evaporated to dryness. The crude product(Suc-VII) was purified on a 4 gram silica gel column eluted with 0-15%MeOH (1% acetic acid) over 15 minutes. Pure fractions were combined andevaporated to provide 190 mg of pure VII-Suc.

Compound VII-Suc (150 mg) was dissolved in 10 mL of DMF and 1 equivalentof HATU was added followed by 2 equivalents of DIPEA. 1.5 equivalents ofglycine-OtBu were added and stirred overnight. The DMF was evaporatedand the residue treated with 5 mL of iN HCl in dioxane for 30 minutes.The solvent was evaporated and the crude Gly-Suc-VII was flash purifiedon a 4 gram silica gel column eluted with 0-10% MeOH over 10 minutes.Evaporation of pure fractions provided 110 mg of Gly-Suc-VII; the purematerial was dissolved in DMF and the above process was repeated toprovide 60 mg of pure Gly2-Suc-VII.

The pure Gly2-Suc-VII (30 mg) was dissolved in 5 mL of DMF and 1.5equivalents of NHS was added followed by 5 mL of THF. DCC (1.5equivalents) was added and the mixture was stirred overnight at roomtemperature. The solvent was evaporated and the crude NHS ester wasflash purified on a silica gel eluted with 0-10% MeOH in DCM over 10minutes. Pure fractions (determined by TLC) were combined and evaporatedto provide 1 mg of pure NHS-Gly2-Suc-VII after lyophilization fromacetonitrile water.

The pure NHS ester was dissolved in DMSO to make a 20 mM solution and 6eq. was added to 2 mL of an IgG antibody (specifically, the anti-CD20antibody rituximab) (10 mg/mL in PBS). The conjugation reaction wasincubated at room temperature overnight and buffer exchanged into freshPBS to remove excess adjuvant. The purified immunoconjugate BB-15 wassterile filtered and stored at 4° C. The yield was about 16 mg. Besideshaving a high yield, the LC/MS analysis showed high levels of purity,low levels of aggregation, and a desirable DAR ratio (see FIGS. 5 and6).

Example 11. Synthesis of Immunoconjugate with a TFP Ester

This example provides guidance on synthesis of an immunoconjugate with adifferent linker using the TFP ester method. Ester modification of theadjuvant and conjugation of the modified adjuvant to the antibody isshown in Scheme 17 of FIG. 143. Compound VII (311 mg, 1 mmol) wasdissolved in 10 mL of DMF and then 0.3 mL of DIPEA was added. TheNHS-PEG5-acid (1.2 equivalents) was dissolved in 5 mL of dichloromethaneand added to compound VII in one portion. The mixture was stirredovernight at room temperature and then concentrated to dryness. Thecrude residue was purified via silica gel chromatography on a 4 gramcolumn eluted with 0-10% MeOH in DCM containing 1% acetic acid over 10minutes to provide 260 mg (57% yield) of PEG5-VII after concentration ofthe pure fractions.

PEG5-VII (50 mg) was dissolved in 10 mL DMF and 1.5 eq. of TFP was addedfollowed by 1.2 eq. DCC and 5 mg of DMAP. The reaction was stirredovernight, concentrated to dryness and purified on silica gel 4 gramcolumn eluted with 0-10% MeOH in DCM to provide 35 mg of pureTFP-PEG5-VII after lyophilization from 1:2 acetonitrile water.

The TFP ester (TFP-PEG5-VII) was dissolved in DMSO to make a 20 mM stocksolution and added to 20 mg of an IgG antibody (specifically, theanti-CD20 antibody rituximab) in PBS at 10 mg/mL. The conjugationreaction was allowed to proceed overnight at room temperature. Theresulting immunoconjugate was buffer exchanged (GE, PD10 desaltingcolumn) into PBS at pH 7.4. The purified immunoconjugate was sterilefiltered using a 2 μm syringe filter and stored at 4° C. LC/MS analysisconfirmed that the process provided a DAR of 2.9 adjuvants per antibody(see FIG. 7). SEC analysis indicated minimal amounts of aggregate (i.e.,less than 2%) (see FIG. 8).

Example 12. Synthesis of Another TLR7/TLR8 Adjuvant

This example provides guidance on how to synthesize another TLR7/8adjuvant. Compound XIV was synthesized starting from compound VI ofScheme 8 of Example 3.

Compound VI (2 g) was dissolved in toluene with 20% dry acetic acid andheated to 75° C. overnight. The solvent was removed under vacuum toprovide 2 grams of crude compound XI. Compound XI was used withoutfurther purification. Compound XI (2 g) was dissolved in 20 mL DMF and1.2 equivalents of NaH (50% dispersion) was added slowly and the mixturewas stirred for 30 minutes at room temperature. Methyl iodide (2equivalents) was added in one portion and the reaction mixture wasstirred overnight at room temperature. The reaction was concentrated todryness and the product purified via flash chromatography. The productwas eluted with a gradient of 0-10% MeOH in dichloromethane over 15 min.Pure fractions were combined and concentrated to yield 1 g of compoundXII (50% yield for 2 steps).

Compound XII (10 g) was dissolved in 10 mL of neat dimethoxybenzylamine(“DMBA”) and heated to 120° C. for 3 hours. The reaction mixture wascooled and diluted with 100 mL of ethyl acetate. The resulting solutionwas washed two times with 10% citric acid in water and once with waterto remove excess DMBA. The organic layer was dried over MgSO4 andconcentrated under vacuum to provide crude compound XIII as a brown oil.The crude DMB derivative, compound XIII, was dissolved indichloromethane and 2 mL of 4N HCl in dioxane was added. After 2 hours,the reaction mixture was concentrated to dryness and the crude HCl saltcompound XIV was dissolved in 3 mL of methanol. Ethyl ether (20 mL) wasadded slowly with stirring to the crude solution and a white precipitateformed. The reaction was filtered and the white solid product was washedtwice with 10 mL ethyl ether and dried under vacuum to provide 4 gram ofHCl salt compound XIV. LC/MS analysis confirmed the correct molecularweight (M/z=326.5) and a purity of greater than 95%.

Example 13. Synthesis of Immunoconjugate BB-26 with a TFP Ester

This example provides guidance on synthesis of an immunoconjugate thatcontains an aryl tertiary amine linker using the TFP ester method asdepicted in Scheme 20 of FIG. 144. Compound XIV (300 mg) of Example 12was dissolved in THF (10 mL) and 1.2 eq. of NaH (50% dispersion) wasadded. The mixture was stirred for 15 minutes and 2 equivalents of4-bromomethylphenyl acetic acid was added. The reaction was stirredovernight at room temperature and concentrated to dryness. One mL ofacetic acid was added and the product was purified by preparative HPLCon a C-18 column eluted with a gradient of 10-90% acetonitrile in water(0.1% TFA) over 20 minutes to provide 165 mg of purified phenylaceticacid compound XV.

Compound XV (50 mg) was dissolved in dichloromethane/dimethylformamide(5 mL, 1:1) and 2 equivalents of TFP was added followed by 1.5equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (“EDCI”).The reaction was stirred overnight at room temperature and the productpurified via flash chromatography on a 4 gram silica gel column elutedwith 0-10% isopropanol over 10 minutes. Pure fractions were concentratedand lyophilized from 30% acetonitrile water to provide 21 mg of purifiedTFP ester compound XVI as a pale yellow solid. The molecular weight andpurity were confirmed by LC/MS (m/z=621.7).

Conjugation to Antibody:

The TFP ester XVI was dissolved in anhydrous DMSO to make a 20 mM stocksolution and 6 molar equivalents (relative to the antibody) was added to20 mg IgG antibody (specifically, the anti-CD20 antibody rituximab) (10mg/mL in PBS). The conjugation reaction was incubated at 4° C.overnight. The resulting immunoconjugate, BB-26, was buffer exchangedinto PBS (pH 7.2) to remove excess small molecular weight reagents. Thefinal concentration was determined by measuring the antibody at 280 nmon the Nanodrop 1000 spectrophotometer. The yield was 15 mg of BB-26, or75% based on recovered protein. As seen in FIG. 12A, minimal aggregatewas seen (less than 1%) as detected by SEC analysis. As seen in FIG.12B, the product had a DAR ratio of 2.8 as determined via LC/MSanalysis. The purified immunoconjugates BB-26 was filtered through a 0.2μM sterile filter and stored at −20° C.

Example 14. Synthesis of Immunoconjugate BB-27 with a TFP Ester

This example provides guidance on synthesis of an immunoconjugate thatcontains an alkyl tertiary amine linker using the TFP ester method asdepicted in Scheme 21 of FIG. 145. Compound XIV (200 mg) was dissolvedin methanol (20 mL) and 3 equivalents of 1-formyl-7-tert-butylheptanoate was added followed by 1.1 equivalents of NaCNBH₄. The mixturewas stirred for 1.5 hours at room temperature and concentrated todryness. TFA (5 mL) was added and the mixture stirred overnight at roomtemperature. The TFA was evaporated under vacuum and the crude productwas purified by preparative HPLC on a C-18 column. The product waseluted with a gradient of 10-90% acetonitrile in water (0.1% TFA) over20 minutes to provide 110 mg of purified acid compound XVII (which wasconfirmed by LC/MS).

Compound XVII (50 mg) was dissolved in dichloromethane/dimethylformamide(5 mL, 1:1) and 2 equivalents of TFP was added followed by 1.5equivalents of EDCI. The reaction was stirred overnight at roomtemperature. The crude TFP ester product XVIII was purified via flashchromatography on a 4 gram silica gel column eluted with 0-10%isopropanol over 10 minutes. Pure fractions were concentrated and theresidue lyophilized from 30% acetonitrile water to provide 14 mg ofpurified TFP ester compound XVIII as a white solid. The molecular weightand purity were confirmed by LC/MS (m/z=601.7).

Conjugation to Antibody:

TFP ester XVIII was dissolved in anhydrous DMSO to make a 20 mM stocksolution and 8 molar equivalents (relative to the antibody) was added to20 mg of an IgG antibody (specifically, the anti-CD20 antibodyrituximab) (10 mg/mL in PBS). The conjugation reaction was incubated at4° C. overnight. The resulting immunoconjugate BB-27 was bufferexchanged into PBS (pH 7.2) to remove excess small molecular weightreagents. The final concentration was determined by measuring theantibodies at 280 nm on the Nanodrop 1000 spectrophotometer. The yieldwas 16 mg of immunoconjugate BB-27 (80%).

Minimal aggregate was seen (less than 1%) as detected by SEC analysis.The product had a DAR ratio of 2.5 as determined via LC/MS analysis. Thepurified BB-27 was filtered through a 0.2 μM sterile filter and storedat −20° C.

Example 15. Synthesis of Immunoconjugate BB-36 with a TFP Ester

This example provides guidance on synthesis of an immunoconjugate thatcontains a PEG tertiary amine linker using the TFP method as depicted inScheme 22 of FIG. 146. Compound XIV (200 mg) was dissolved in methanol(20 mL) and 3 eq. of aldehyde XIX was added followed by 1.1 equivalentsof NaCNBH₄. The mixture was stirred for 3 hours at room temperature andconcentrated to dryness. Trifluoroacetic acid (TFA, 10 mL) was added andthe reaction stirred for 2 hours at room temperature. The TFA wasevaporated under vacuum and the crude product was purified bypreparative HPLC on a C-18 column. The product was eluted with agradient of 10-90% acetonitrile in water (0.1% TFA) over 20 minutes toprovide 85 mg of purified acid XX after lyophilization of the combinedpure fractions (confirmed by LC/MS).

Compound XX (80 mg) was dissolved in dichloromethane/dimethylformamide(5 mL, 1:1) and 2 equivalents of TFP was added followed by 1.2equivalents of EDCI. The reaction was stirred overnight at roomtemperature. The crude TFP ester product XXI was purified via flashchromatography on a 4 gram silica gel column eluted with 0-10%isopropanol over 10 minutes. Pure fractions were concentrated and theresidue lyophilized from 30% acetonitrile water to provide 45 mg ofpurified TFP ester of compound XXI as a beige solid. The molecularweight and purity were confirmed by LC/MS (m/z=647.7).

Conjugation to Antibody:

The TFP ester of compound XXI was dissolved in anhydrous DMSO to make a20 mM stock solution and 8 molar equivalents (relative to the antibody)was added to an IgG1 antibody (specifically, the anti-CD20 antibodyrituxumab) (10 mg/mL in PBS). The conjugation reaction was incubated at4° C. overnight. The resulting immunoconjugate BB-36 was bufferexchanged into PBS (pH 7.2) to remove excess small molecular weightreagents. The final concentration was determined by measuring theantibodies at 280 nm on the Nanodrop 1000 spectrophotometer. The yieldwas 15 mg of immunoconjugate BB-36 (75%) which was stored at 4° C. untilused.

Minimal aggregate was seen (less than 1%) as detected by SEC analysis.The product had a DAR ratio of 2.2 as determined via LC/MS analysis. Thepurified immunoconjugate BB-36 was filtered through a 0.2 μM sterilefilter and stored at −20° C.

Example 16. Synthesis of Immunoconjugate BB-45 with a TFP Ester

This example provides guidance on synthesis of an immunoconjugate with adifferent linker using the TFP ester method as depicted in Scheme 23 ofFIG. 147. Compound VII (311 mg, 1 mmol) was dissolved in 10 mL of DMFand 0.3 mL of DIPEA was added. In a separate container, 1.2 equivalentsof 7-methoxy-7-oxoheptanoic acid was dissolved in 5 mL of DMF and 1.5equivalents DIPEA was added followed by HATU (1.2 equivalents). Themixture was added to VII and stirred overnight at room temperature. Thereaction mixture was concentrated to dryness under vacuum and theresidue was dissolved in 10 mL of (1:1) tetrahydrofuran:water. One mL of2M lithium hydroxide in water was added and the reaction stirred for 2hours at room temperature. The THF was removed via rotary evaporationand the aqueous solution was acidified by adding 10 mL of 1Mhydrochloric acid. The aqueous solution was extracted 2x withdichloromethane (20 mL) and the organic layer was combined and driedover magnesium sulfate. The solution was filtered and the filtrateconcentrated to dryness. The crude product 22 was purified via silicagel chromatography on a 4 gram column eluted with 0-10% isopropanol inDCM (w/1% acetic acid) over 10 minutes. The pure fractions were combinedand concentrated to provide 220 mg of pure 22 as a pale yellow solid.

Compound 22 (50 mg) was dissolved in dichloromethane/dimethylformamide(5 mL, 1:1) and 2 equivalents of TFP was added followed by 1.5equivalents of EDCI. The reaction was stirred overnight at 22° C. andthe crude reaction was concentrated to dryness. The product was purifiedvia flash chromatography on a 4 gram silica gel column eluted with 0-10%isopropanol over 10 minutes. Pure fractions were concentrated and theresidue was lyophilized from 30% acetonitrile in water to provide 21 mgof purified TFP ester 23 as a pale yellow solid. The molecular weightand purity were confirmed by LC/MS.

Conjugation to Antibody:

The TFP ester 23 was dissolved in anhydrous DMSO to make a 20 mM stocksolution and 6 molar equivalents (relative to the antibody) was added to20 mg of an IgG antibody (specifically, the anti-CD20 antibodyrituximab) (10 mg/mL in PBS). The conjugation reaction was incubated at4° C. overnight. The resulting immunoconjugate BB-45 was bufferexchanged into PBS (pH 7.2) to remove excess small molecular weightimpurities. The final concentration was determined by measuring theabsorbance at 280 nm on a Thermo Nanodrop 1000 spectrophotometer. Theyield was 14 mg of BB-45, or 70% based on recovered protein. Minimalaggregate (less than 1%) was detected by SEC analysis and a DAR of 2.8was determined via LC/MS analysis. The purified immunoconjugate wasfiltered through a 0.2 μM sterile filter and stored at −20° C.

Example 17. Synthesis of Immunoconjugate BB-24 with a TFP Ester

This example provides guidance on synthesis of an immunoconjugate with adifferent linker using the TFP ester method as depicted in Scheme 24 ofFIG. 148. Compound VII (150 mg) was dissolved in 20 mL THF and 10 mL ofaqueous saturated sodium bicarbonate was added. Succinic anhydride (50mg) was added in one portion and the mixture stirred for 1 hour at roomtemperature. 20 mL of 1N HCl was added slowly and the mixture wasextracted with 2X 50 mL of dichloromethane and the combined organicextracts were evaporated to dryness. The crude product 24 was purifiedon a 4 gram silica gel column eluted with 0-15% MeOH (1% acetic acid)over 15 minutes. Pure fractions were combined and evaporated to provide180 mg of pure 24.

One hundred and fifty mg of 24 was dissolved in DMF (10 mL) and 1equivalent of HATU was added followed by 2 equivalents of DIPEA. One anda half eq. of glycine-OtBu was added and stirred overnight. The DMF wasevaporated and the residue treated with 5 mL of iN HCl in dioxane for 30minutes with stirring. The solvent was evaporated and the crude residuewas flash purified on a 4 gram silica gel column eluted with 0-10%isopropanol over 15 minutes. Evaporation of pure fractions provided 110mg of pure 25.

Compound 25 (50 mg) was dissolved in 10 mL DMF and 1.5 eq. of TFP wasadded followed by 1.2 eq. DCC and 2 mg of DMAP. The reaction was stirredovernight, concentrated to dryness and purified on silica gel (4gcolumn) eluted with 0-10% IPA in DCM to provide 32 mg of pure TFP ester,compound 26, after lyophilization from 1:3 acetonitrile water.

Conjugation to Antibody:

The TFP ester, compound 26, was dissolved in anhydrous DMSO to make a 20mM stock solution and 5 molar equivalents (relative to the antibody) wasadded to 20 mg antibody at 10 mg/mL in PBS. The conjugation reaction wasincubated at 4° C. for 6 hours. The resulting immunoconjugate BB-24 wasbuffer exchanged into PBS (pH 7.4) to remove excess small molecularweight impurities. The final protein concentration was determined bymeasuring the absorbance at 280 nm on a Nanodrop 1000 spectrophotometer.The yield was 15 mg (75% based on recovered protein). SEC analysisdetected minimal aggregate of less than 1% and the DAR was determined tobe 2.8 adjuvants per antibody via LC/MS analysis. The purifiedimmunoconjugate was filtered through a 0.2 μM sterile filter and storedat −20 OC until needed.

Example 18. Synthesis of Immunoconjugate BB-37 a TFP Ester

This example provides guidance on synthesis of an immunoconjugate with adifferent linker using the TFP method as depicted in Scheme 25 of FIG.149. Compound VII (155 mg, 0.5 mmol) was dissolved in 10 mL of DMF and0.2 mL of DIPEA was added. In a separate container, 1.2 equivalents ofPEG2-dicarboxylate mono methyl ester was dissolved in 5 mL of DMF and 2equivalents DIPEA was added followed by HATU (1.2 equivalents). Themixture was added to VII and stirred 1 hour at room temperature. Thereaction was concentrated to dryness under vacuum and the residue wasdissolved in THF (5 mL). An equal volume of water was added followed by2 mL of 1 M aqueous LiOH. The mixture was stirred overnight and then 10mL of 1N HCl was added. The acidified mixture was extracted 2x withdichloromethane, dried over sodium sulfate, concentrated to dryness andpurified via silica gel chromatography. The product was eluted with0-10% methanol over 10 minutes. The pure fractions were combined andconcentrated to provide 110 mg of pure compound 27 as a pale yellowsolid.

Compound 27 (50 mg) was dissolved in dichloromethane/dimethylformamide(5 mL, 1:1) and 2 equivalents of TFP was added followed by 1.5equivalents of EDCI. The reaction was stirred overnight at ambienttemperature and the reaction was concentrated to dryness. The crude TFPester 28 was purified via flash chromatography on a 4 gram silica gelcolumn eluted with 0-10% isopropanol over 10 minutes. Pure fractionswere concentrated and the residue was lyophilized from 30% acetonitrilein water to provide 41 mg of purified TFP ester 23 as a white solid. Themolecular weight and purity were confirmed by LC/MS.

Conjugation to Antibody:

The TFP ester 28 was dissolved in anhydrous DMSO to make a 20 mM stocksolution and 8 molar equivalents (relative to the antibody) was added to20 mL of an IgG antibody (specifically, the anti-CD20 antibodyrituximab) (10 mg/mL in PBS). The conjugation reaction was incubated at4° C. overnight. The resulting immunoconjugate BB-37 was bufferexchanged into PBS (pH 7.2) to remove excess small molecular weightimpurities. The final concentration was determined by measuring theabsorbance at 280 nm on a Thermo Nanodrop 1000 spectrophotometer. Theyield was 16 mg of conjugated immunoconjugate BB-37, or 70% based onrecovered protein. Minimal aggregate (less than 1%) was detected by SECanalysis and a DAR of 2.3 was determined via LC/MS analysis. Thepurified immunoconjugate was filtered through a 0.2 M sterile filter andstored at −20° C.

Example 19. Synthesis of Another TLR7/8 Adjuvant

This example provides guidance on synthesis of another TLR agonist.Compound 29 is a compound VII analog that contains a piperizineside-chain for linker attachment. It was synthesized using methodspreviously described for the synthesis of the compound VII except that aBoc-protected piperizine analog was substituted for Boc-diaminobutaneused in step 3 of the synthesis. The general synthetic route forcompound 29 is outlined in Scheme 26. The addition of the piperizineside chain enables the synthesis of immunoconjugates that werepreviously inaccessible due to instability. Similar compound VII analogscontaining succinate linkers are prone to cyclization upon TFPactivation and the piperizine prevents cyclization. In addition, thetertiary amino group within the piperizine moeity maintains a positivecharge after linker attachment and conjugation. Positive charges in thislocation are important for improved TLR8 potency. Compound 29 wassubsequently used for synthesizing immunoconjugates as described belowin Examples 19-21.

Example 20. Synthesis of Immunoconjugate BB-42 with a TFP Ester

This example provides guidance on synthesis of an immunoconjugate with adifferent linker using the TFP ester method as depicted in Scheme 27 ofFIG. 15O. Compound 29 (100 mg) was dissolved in 10 mL THF and 2 mL ofaqueous saturated sodium bicarbonate was added followed by 10 mL ofwater. Succinic anhydride (50 mg) was added in one portion and themixture was stirred at room temperature. After one hour, 20 mL of iN HClwas added slowly and the reaction mixture was extracted with 2X 50 mL ofdichloromethane (“DCM”). The combined organic extracts were evaporatedto dryness. The crude product 30 was purified on a 4 gram silica gelcolumn eluted with 0-15% isopropanol in DCM (1% acetic acid) over 15minutes. Pure fractions were combined and evaporated to dryness toprovide 80 mg of pure acid 30.

Compound 30 (50 mg) was dissolved in dichloromethane/dimethylformamide(5 mL, 1:1) and 2 equivalents of TFP was added followed by 1.5equivalents of EDCI. The reaction was stirred overnight at ambienttemperature and the reaction was concentrated to dryness. The crude TFPester 31 was purified via flash chromatography and eluted with 0-10%isopropanol over 10 minutes. Pure fractions were concentrated and theresidue was lyophilized from 30% acetonitrile in water to provide 41 mgof purified TFP ester 31 as a white solid. The molecular weight andpurity were confirmed by LC/MS.

The TFP ester 31 was conjugated to an IgG1 antibody (specifically, theanti-CD20 antibody rituxumab) as described previously for BB-24 toprovide BB-42. SEC and LC/MS analysis of BB-42 confirmed the molecularweight, a high monomeric purity with less than 2% aggregate, and a DARof 1.7 (see FIGS. 20A-B).

Example 21. Synthesis of Immunoconjugates BB-43 and BB-44 with a TFPEster

This example provides guidance on synthesis of immunoconjugates withdifferent linkers using the TFP ester method as depicted in Scheme 28 ofFIG. 15I. Compound 30 (Scheme 27) was coupled to polyethylene glycol(PEG) linkers containing 2 or 8 PEG. units in order to extend thedistance between the adjuvant and the antibody. Attachment of the PEGlinker extensions was performed using previously described protocols forlinker attachment and TFP activation. Briefly 100 mg of compound 30 wasdissolved in 10 mL of DMF and 0.2 mL of DIPEA was added followed by HATU(1.2 equivalents). After 1 hour the appropriate amino PEG linker (n=2 or8) was added and stirred an additional 2 hours at room temperature. Thereaction mixture was concentrated to dryness under vacuum and theresidue was purified via preparative HPLC on a C-18 column eluted with10-90% acetonitrile in water over 30 minutes. The pure fractions werecombined and lyophilized to provide 65 mg and 45 mg of intermediates 31or 32 as a clear glassy substance.

Compounds 31 and 32 were converted to the corresponding TFP esters 33and 34 using previously described protocols. Briefly, the free acid 31or 32 (50 mg) was dissolved in dichloromethane/dimethylformamide (5 mL,1:1) and 2 equivalents of TFP was added followed by 1.5 equivalents ofEDCI. The mixture was stirred overnight at room temperature andconcentrated to dryness to provide crude TFP esters 33 and 34. The crudeTFP esters were purified via flash chromatography on silica gel andeluted with 0-10% isopropanol over 10 minutes. Pure fractions wereconcentrated and the residue was lyophilized from 30% acetonitrile inwater to provide purified TFP esters 33 and 34 as clear solids. Themolecular weight and purity of the pure compounds were confirmed byLC/MS.

Conjugation to Antibody:

TFP esters 33 and 34 were conjugated to an IgG1 antibody (specifically,the anti-CD20 antibody rituxumab) using previously described protocols.The TFP esters were dissolved in anhydrous DMSO to make a 20 mM stocksolution and 8 molar equivalents (relative to the antibody) was added to20 mg of the IgG antibody at 10 mg/mL in PBS. The conjugation reactionwas incubated at 4° C. for 12 hours. The resulting immunoconjugates,BB-43 and BB-44 were buffer exchanged into PBS (pH 7.4) to remove excesssmall molecular weight impurities. The final protein concentration wasdetermined by measuring the absorbance at 280 nm on a Nanodrop 1000spectrophotometer. The yields were 75% based on recovered protein. SECanalysis detected minimal aggregate was present and the DARs of 1.0 and1.7 adjuvants per antibody were determined via LC/MS analysis. Thepurified immunoconjugates were filtered through a 0.2 M sterile filterand stored at −20 OC until needed.

Example 22. Assessment of Immunoconjugate Activity In Vitro

Isolation of Human Antigen Presenting Cells.

Human antigen presenting cells (APCs) were negatively selected fromhuman peripheral blood mononuclear cells obtained from healthy blooddonors (Stanford Blood Center) by density gradient centrifugation usinga RosetteSep Human Monocyte Enrichment Cocktail (Stem Cell Technologies)containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123,and HLA-DR. Immature APCs were subsequently purified to >97% purity vianegative selection using an EasySep Human Monocyte Enrichment Kitwithout CD 16 depletion containing monoclonal antibodies against CD14,CD16, CD40, CD86, CD123, and HLA-DR.

Preparation of Tumor Cells.

Tumor cells were resuspended in PBS with 0.1% fetal bovine serum (FBS)at 1 to 10×10⁶ cells/mL. Cells were subsequently incubated with 2 M CFSEto yield a final concentration of 1 μM. The reaction was ended after 2minutes via the addition of 10 mL complete medium with 10% FBS andwashed once with complete medium. Cells were either fixed in 2%paraformaldehyde and washed three times with PBS or left unfixed priorto freezing the cells in 10% DMSO, 20% FBS and 70% medium.

APC-Tumor Co-Cultures.

2×10⁵ APCs were incubated with or without 6.5×10⁵ allogeneicCFSE-labeled tumor cells in 96-well plates (Corning) containing IMDMmedium (Gibco) supplemented with 10% fetal bovine serum, 100 U/mLpenicillin, 100 g/mL streptomycin, 2 mM L-glutamine, sodium pyruvate,non-essential amino acids and, where indicated, various concentrationsof unconjugated CD20 antibody, and immunoconjugates of the inventionwere prepared according to the examples above. Cells and cell-freesupernatants were analyzed after 18 hours via flow cytometry or ELISA.

The results of this assay are shown in FIGS. 9A-9F for BB-17 and BB-01.Specifically, the graphs show that BB-17 and BB-01 prepared according toSchemes 14 of FIG. 140 and 17 of FIG. 143 elicits myeloid activationwhile the control, unconjugated CD20 antibody, does not. Further, FIGS.23A-D show that BB-14 elicits myeloid activation as indicated by CD14,CD20, CD86, and HLA-DR while the control does not. FIGS. 24A-D show thatBB-15 elicits myeloid activation as indicated by CD14, CD20, CD86, andHLA-DR while the control does not. FIGS. 25A-D show that BB-27 elicitsmyeloid activation as indicated by CD14, CD20, CD86, and HLA-DR whilethe control does not. FIGS. 26A-D show that BB-45 elicits myeloidactivation as indicated by CD14, CD20, CD86, and HLA-DR while thecontrol does not. FIGS. 27A-D show that BB-24 elicits myeloid activationas indicated by CD14, CD20, CD86, and HLA-DR while the control does not.

Example 23. Comparison of BB-01 to Comparative Conjugate IRM1 andComparative Conjugate IRM2

As previously explained, immunoconjugates are described in U.S. Pat. No.8,951,528 (“the '528 patent”). This example shows that immunoconjugatesof the invention are superior to the immunoconjugates disclosed by the'528 patent. BB-01 was synthesized according to Scheme 14 of FIG. 140.Comparative Conjugates IRM1 and IRM2 were prepared using the adjuvantsdescribed in the '528 patent as adjuvants IRM1 and IRM2. Specifically,IRM1 and IRM2 were conjugated to an IgG antibody (specifically, theanti-CD20 antibody rituxumab) with an amide linker.

BB-01 and Comparative Conjugates IRM1 and IRM2 were analyzed using theassay of Example 22. The results are shown in FIGS. 10A-10F and 11A-11C.Specifically, FIGS. 10A-10F show that BB-01 prepared according to Scheme14 of FIG. 14O elicits myeloid activation while Comparative ConjugatesIRM1 and IRM2, and the control, unconjugated CD20 antibody, do not.Further, FIGS. 11A-11C show that BB-01 prepared according to Scheme 14of FIG. 14O elicits cytokine secretion while Comparative Conjugates IRM1and IRM2, and the control, unconjugated CD20 antibody, do not.

The Comparative Conjugates IRM1 and IRM2 had excessive aggregation asdetermined by LC/MS. FIGS. 15A-C show the results of size exclusionchromatography following filtration with a 0.2 M filter. ComparativeConjugate IRM1 had 4% aggregation and indicated by the first peak at 4.5min. Comparative Conjugate IRM2 had 9.5% aggregation and indicated bythe first peak at 4.5 min. In contrast, BB-01 had a small amount ofaggregation. This difference is due in part to the thiolatedintermediate that IRM1 and IRM2 have which is not necessary for thesynthesis of BB-01.

BB-01 and Comparative Conjugates IRM1 and IRM2 were also tested forstorage stability. After synthesis, the conjugates were stored in 15 mLconical tubes for several hours. After storage, the tube containing theComparative Conjugate IRM2 had a large white solid aggregate at thebottom of the tube. The tubes containing BB-01 and Comparative ConjugateIRM1 contained clear fluid only and did not have any sediment.

Example 24. Generation of Anti-Compound 1 Antibody

KLH (ThermoFisher, Product #77600) or Bovine Serum Albumin (ThermoFisher, Product #29130) was conjugated to Compound 1 usingamine-reactive chemistry.

To produce rabbit antibodies, rabbits were immunized by injecting thefootpad with 200 ug of KLH-Compound 1 conjugate, formulated in CompleteFreund's adjuvant. Animals were boosted with an additional 100 ug ofimmunogen conjugate 14, 28, and 42 days following the firstadministration. Blood was collected on days 35 and 49 and serum wasisolated and screened by ELISA for anti-Compound 1 antibody. ELISAplates were coated with BSA-Compound 1 conjugate and antibodies weredetected with peroxidase-conjugated anti-rabbit IgG (JacksonImmunoresearch, Product #111-035-144).

To produce murine antibodies, C57BL/6 mice were injected intravenouslywith 100 ug of Compound 1 conjugate, followed by repeat doses at days 6,12 and 24 post first administration. Blood was collected 12 and 24 dayspost administration and serum was screened by ELISA for anti-Compound 1antibody. Following sufficient detection of antibody, blood, spleen andlymph nodes were collected and harvested into single cell suspension. Bcells were subsequently isolated by negative selection and sorted usingFACS. B cells were collected that stained positive for IgG, stainednegative for IgM and IgD, and stained positive for Compound 1engagement, as measured using a BSA-Compound 1 conjugate andfluorescently labeled Streptavidin. Isolated B cells were washed twicein complete medium and then fused with SP20 myeloma cells usingpolyethylene glycol 1500 (Roche, Product #10 783 641 001) according tomanufacturer's instructions. SP20 myeloma cells were maintained prior tofusion in DMEM supplemented with 10% FBS, Glutamine and PenicillinStreptomycin. Fused cells were plated at approximately 100,000 cells perwell in flat 96 well plates. Following 1-2 days of incubation, HATsupplement and IL-6 were added to the medium (ThermoFisher Product#21060017 and Gibco Product # PHC0065). Medium was sampled 10-14 dayslater and screened by ELISA as described previously to measureanti-Compound 1 antibody. Positive clones were expanded, sub-cloned bylimiting dilution, and were further screened to confirm antibodyproduction and hybridomas were subsequently cryopreserved. Hybridomaswere grown in tissue culture treated flasks at 37 degrees Celsius with5% CO₂ in 10% complete medium and 90% Hybridoma-SFM (Gibco, Product#12045076). Medium was replaced with 100% Hybridoma-SFM and cells werecultured for an additional 3-6 days. Medium was collected and filteredthrough a 0.22 um filter. Antibody was purified using Hi-Trap MabselectColumns (GE Life Sciences, Product #28-4082-53) and buffer exchangedinto sterile PBS by dialysis or through desalting columns.

Example 25: Detection of Conjugation Via ELISA

In five cases, the conjugation status of an antibody construct could notbe resolved through LC/MS due to product heterogeneity. In order todetermine if the conjugation was successful, an ELISA assay wasutilized. The antibody used to detect presence of adjuvant on theantibody was the anti-Compound 1 antibody described in Example 24.

FIGS. 132A-132H indicate that the conjugations were successful for thecetuximab immunoconjugate, etanercept naked antibody, etanerceptimmunoconjugate, ipilimumbab immunoconjugate, and obinutuzumabimmunoconjugate.

Example 26. ELISA Detection of Compound 1 Coupled to Human IgG of HumanIg-Fc

Maxysorp ELISA plates (Fisher 44-2404-21) were coated overnight with 1ug/ml Goat anti-human IgG (Jackson Immunoresearch). Plates were blockedwith PBS containing 1% BSA (Sigma A7030), and incubated with a titrationof the indicated antibodies or corresponding Boltbody (BB-01)conjugates. Bound antibodies were detected with Peroxidase conjugatedGoat Anti-Human IgG (Jackson), or a mouse monoclonal antibody againstCompound 1 followed by Peroxidase conjugated Goat anti-mouse IgG (Fcfragment specific). TMB was added to the wells and absorbance at 450 nMwas measured after stopping the reaction with TMB stop solution (FisherNC1291012).

Example 27. ELISA Detection of Compound 1 Coupled to Rat-Anti-Dectin 2

Maxisorp ELISA plates (Fisher 44-2404-21) were coated overnight with 1ug/ml Rat anti-Dectin-2 (Invivogen) or BB-01 Rat anti-Dectin-2. Plateswere blocked with PBS containing 1% BSA (Sigma A7030), and incubatedwith titrating amounts of peroxidase conjugated Goat anti-mouse IgG,heavy and light chain specific (Jackson 115-035-003) for total IgGdetection, or titrating amounts of rabbit anti-Compound 1 antiserum forBoltbody detection. Rabbit anti-Compound 1 was detected with peroxidaseconjugated Goat anti rabbit IgG, minimal cross reactivity with human,mouse and Rat serum proteins (Jackson 111-035-144). TMB was added to thewells and absorbance at 450 nM was measured after stopping the reactionwith TMB stop solution (Fisher NC1291012).

Although the foregoing has been described in some detail by way ofillustration and example for purposes of clarity and understanding, oneof skill in the art will appreciate that certain changes andmodifications can be practiced within the scope of the appended claims.In addition, each reference provided herein is incorporated by referencein its entirety to the same extent as if each reference was individuallyincorporated by reference.

Example 28. Method for Determining Protein A Binding Activity

Duplicate samples of Rituximab or Rituximab BB-37 (100 ul, 50 ug/ml inPBS) were incubated with 12.5 ul protein A sepharose beads (ThermoFisher 22810) with rotating overnight. Beads were pelleted bycentrifugation, supernatant was removed, and residual liquid was removedfrom the beads using a fine pipette tip. Non-reducing Laemmli samplebuffer (100 ul) was added to the beads. Beads and supernatants wereheated to 90° C. for 5 minutes and equal fractions were analyzed bySDS-PAGE (4-12% NUPAGE gel, MOPS buffer) followed by staining withCoomassie (GelCode™ Blue, ThermoFisher). Molecular weight standard isSeeBlue® Plus 2 marker (ThermoFisher LC5925). As seen in FIG. 133C,preservation of protein A binding in Rituximab BB-37 suggestspreservation of FcRN binding.

Example 29. Method for Determination of Binding Activity to CD16a

Maxysorp ELISA plates were coated overnight with 1.5 ug/ml recombinanthuman CD16a protein (R&D Systems 4325-FC-050). Plates were blocked withPBS containing 1% BSA, and incubated with a titration of antibodies orantibody immunoconjugates. Bound antibodies were detected withPeroxidase conjugated AffiniPure F(ab′)₂ Fragment Goat Anti-Human IgG(Jackson 109-036-003). TMB (Fisher PI34028) was added to the wells andabsorbance at 450 nM was measured after stopping the reaction with TMBstop solution (Fisher NC1291012). As seen in FIG. 133A, the aglycosylmutant of Rituximab shows diminished binding, consistent with the roleof glycosylation in effector function.

Example 30. Method for Determination of Binding Activity to CD64

Maxysorp ELISA plates were coated overnight with 1.5 ug/ml recombinanthuman CD64 protein (R&D Systems). Plates were blocked with PBScontaining 1% BSA, and incubated with a titration of Rituximab orRituximab immunoconjugates (Rituximab BB-01). Bound antibodies weredetected with Peroxidase conjugated AffiniPure F(ab′)₂ Fragment GoatAnti-Human IgG (Jackson) using TMB color development and absorbance at450 nM was measured after stopping the reaction. As seen in FIG. 133B,Rituximab had been deglycosylated used PNGase F shows impaired bindingto CD64.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. An immunoconjugate comprising (a) an antibody construct comprising(i) an antigen binding domain and (ii) an Fc domain, (b) an adjuvantmoiety of formula:

wherein each J independently is hydrogen, OR⁴, or R⁴, each R⁴independently is hydrogen, or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 carbons, each U independently is CH or N whereinat least one U is N, each subscript t independently is an integer from 1to 3, Q is optionally present and is an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 carbons, and the dashed line (“

”) represents the point of attachment of the adjuvant, and (c) a linker,wherein the adjuvant moiety is covalently bonded to the antibodyconstruct via the linker.
 2. The immunoconjugate of claim 1, wherein theantigen binding domain binds to an antigen selected from the groupconsisting of CDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM,MUC1, MUC16, EGFR, VEGF, HER2, SLAMF7, PDGFRa, gp75, CTLA4, PD-1, PD-L1,PD-L2, LAG-3, B7-H4, KIR, TNFRSF4, OX40L, IDO-1, IDO-2, CEACAMi, BTLA,TIM3, A2Ar, VISTA, CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL,CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5),CLECiB (CLEC-2), CLEC9A (DNGR-1), and CLEC7A (Dectin-1).
 3. Theimmunoconjugate of claim 1, wherein the antigen binding domain binds toEGFR, HER2, or PD-L1.
 4. The immunoconjugate of claim 1, wherein theantibody construct is an antibody.
 5. The immunoconjugate of claim 4,wherein the antibody is selected from the group consisting ofpembrolizumab, nivolumab, atezolizumab, avelumab, ipilimumab,obinutuzumab, trastuzumab, cetuximab, rituximab, pertuzumab,bevacizumab, daratumumab, etanercept, olaratumab, elotuzumab,margetuximab, and a biosimilar thereof.
 6. The immunoconjugate of claim4, wherein the antibody comprises a modified Fc region.
 7. Theimmunoconjugate of claim 4, wherein immunoconjugate has a structureaccording to Formula I:

or a pharmaceutically acceptable salt thereof, wherein Ab is theantibody with

 being an amino acid residue of the antibody, wherein A is a modified orunmodified sidechain of the amino acid residue, Z is the linker, Adj isthe adjuvant moiety, and subscript r is an integer from 1 to
 10. 8. Theimmunoconjugate of claim 7, wherein the immunoconjugate has a structureaccording to Formula II:

or a pharmaceutically acceptable salt thereof, wherein Ab is theantibody with

 being an amino acid residue of the antibody, wherein A is a lysinesidechain, thiol-modified lysine sidechain, or cysteine sidechain of theamino acid residue, Adj is the adjuvant moiety, subscript r is aninteger 1 to 10, Z¹ is selected from —C(O)—, —C(O)NH—, and —CH₂—, Z² andZ⁴ are each independently selected from a bond, C₁₋₃₀ alkylene, and 3-to 30-membered heteroalkylene, wherein one or more groupings of adjacentatoms in the C₁₋₃₀ alkylene and 3- to 30-membered heteroalkylene areoptionally and independently replaced by —C(O)—, —NR^(a)C(O)-, or—C(O)NR^(a)-, wherein each R^(a) is independently selected from H andC₁₋₆ alkyl, one or more groupings of adjacent atoms in the C₁₋₃₀alkylene and 3- to 30-membered heteroalkylene are optionally andindependently replaced by a 4- to 8-membered divalent carbocycle, andone or more groupings of adjacent atoms in the C₁₋₃₀ alkylene and 3- to30-membered heteroalkylene are optionally and independently replaced bya 4- to 8-membered divalent heterocycle having one to four heteroatomsselected from O, S, and N, Z³ is a bond, a divalent peptide moiety, or adivalent polymer moiety, and Z⁵ is selected from an amine-bonded moietyand a thiol-bonded moiety.
 9. The immunoconjugate of claim 4, whereinthe immunoconjugate has a structure according to Formula III:

or a pharmaceutically acceptable salt thereof, wherein Ab is theantibody with

 being a lysine residue of the antibody, Adj is the adjuvant moiety, Gis CH₂, C═O, or a bond, L is a linking moiety, and subscript r is aninteger from 1 to
 10. 10. The immunoconjugate of claim 9, wherein L isselected from:

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbons, a is an integer from 1to 40, each A is independently selected from any amino acid, subscript cis an integer from 1 to 20, the dashed line (“

”) represents the point of attachment to

and the wavy line (“

”) represents the point of attachment to


11. An immunoconjugate comprising (a) an antibody construct comprising(i) an antigen binding domain and (ii) an Fc domain, (b) an adjuvantmoiety of formula:

wherein each J independently is hydrogen, OR⁴, or R⁴, each R⁴independently is hydrogen, or an alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl groupcomprising from 1 to 8 carbons, Q is optionally present and is an alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl,or heteroarylalkyl group comprising from 1 to 8 carbons, and the dashedline (“

”) represents the point of attachment of the adjuvant, and (c) a linker,wherein the adjuvant moiety is covalently bonded to the antibodyconstruct via the linker.
 12. The immunoconjugate of claim 11, whereinthe antigen binding domain binds to an antigen selected from the groupconsisting of CDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM,MUC1, MUC16, EGFR, VEGF, HER2, SLAMF7, PDGFRa, gp75, CTLA4, PD-1, PD-L1,PD-L2, LAG-3, B7-H4, KIR, TNFRSF4, OX40L, IDO-1, IDO-2, CEACAMi, BTLA,TIM3, A2Ar, VISTA, CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL,CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5),CLECiB (CLEC-2), CLEC9A (DNGR-1), and CLEC7A (Dectin-1).
 13. Theimmunoconjugate of claim 11, wherein the antigen binding domain binds toEGFR, HER2, or PD-L1.
 14. The immunoconjugate of claim 11, wherein theantibody construct is an antibody.
 15. The immunoconjugate of claim 14,wherein the antibody is selected from the group consisting ofpembrolizumab, nivolumab, atezolizumab, avelumab, ipilimumab,obinutuzumab, trastuzumab, cetuximab, rituximab, pertuzumab,bevacizumab, daratumumab, etanercept, olaratumab, elotuzumab,margetuximab, and a biosimilar thereof.
 16. The immunoconjugate of claim14, wherein the antibody comprises a modified Fc region.
 17. Theimmunoconjugate of claim 14, wherein immunoconjugate has a structureaccording to Formula I:

or a pharmaceutically acceptable salt thereof, wherein Ab is theantibody with

 being an amino acid residue of the antibody, wherein A is a modified orunmodified sidechain of the amino acid residue, Z is the linker, Adj isthe adjuvant moiety, and subscript r is an integer from 1 to
 10. 18. Theimmunoconjugate of claim 17, wherein the immunoconjugate has a structureaccording to Formula II:

or a pharmaceutically acceptable salt thereof, wherein Ab is theantibody with

 being an amino acid residue of the antibody, wherein A is a lysinesidechain, thiol-modified lysine sidechain, or cysteine sidechain of theamino acid residue, Adj is the adjuvant moiety, subscript r is aninteger 1 to 10, Z¹ is selected from —C(O)—, —C(O)NH—, and —CH₂—; Z² andZ⁴ are each independently selected from a bond, C₁₋₃₀ alkylene, and 3-to 30-membered heteroalkylene, wherein one or more groupings of adjacentatoms in the C₁₋₃₀ alkylene and 3- to 30-membered heteroalkylene areoptionally and independently replaced by —C(O)—, —NR^(a)C(O)—, or—C(O)NR^(a)-, wherein each R^(a) is independently selected from H andC₁₋₆ alkyl, one or more groupings of adjacent atoms in the C₁₋₃₀alkylene and 3- to 30-membered heteroalkylene are optionally andindependently replaced by a 4- to 8-membered divalent carbocycle, andone or more groupings of adjacent atoms in the C₁₋₃₀ alkylene and 3- to30-membered heteroalkylene are optionally and independently replaced bya 4- to 8-membered divalent heterocycle having one to four heteroatomsselected from O, S, and N; Z³ is a bond, a divalent peptide moiety, or adivalent polymer moiety; and Z⁵ is selected from an amine-bonded moietyand a thiol-bonded moiety.
 19. The immunoconjugate of claim 14, whereinthe immunoconjugate has a structure according to Formula III:

or a pharmaceutically acceptable salt thereof, wherein Ab is theantibody with

 being a lysine residue of the antibody, Adj is the adjuvant moiety, Gis CH₂, C═O, or a bond, L is a linking moiety, and subscript r is aninteger from 1 to
 10. 20. The immunoconjugate of claim 19, wherein L isselected from:

wherein R is optionally present and is a linear or branched, cyclic orstraight, saturated or unsaturated alkyl, heteroalkyl, aryl, orheteroaryl chain comprising from 1 to 8 carbons, a is an integer from 1to 40, each A is independently selected from any amino acid, subscript cis an integer from 1 to 20, the dashed line (“

”) reprepresents the point of attachment to

and the wavy line (“

”) represents the point of attachment to